BR102018011804A2 - implant, and methods for preparing an implant and treating a bone defect. - Google Patents

Abstract

An implant configured to fit in or near a bone defect to promote bone growth is provided. The implant comprises an oxygen uniformly disposed in an acellular tissue matrix (atm). The matrix of the acellular tissue may be porcine collagen, which in some cases is cross-linked. The implant may contain an acellular porcine cross-linked collagen in an amount from about 5 wt% to about 25 wt% of the implant and an oxyesterol in an amount from about 5 wt% to about 90 wt% of the implant. implantation. the oxygen may be oxy133 monohydrate or an oxy133 polymorph. Implant preparation and use methods are provided.

Description

“IMPLANT, AND, METHODS TO PREPARE AN IMPLANT AND TO TREAT A BONE DEFECT”

BACKGROUND [001] Biological products are commonly used to promote bone growth in medical applications, including fracture healing and surgical control of spinal disorders. Spinal fusion is often performed by orthopedic surgeons and neurosurgeons to treat degenerative diseases of the disc and arthritis that affect the lumbar and cervical spine, to correct deformities caused by scoliosis and to repair instability due to spondylolisthesis. In addition, spinal fusion techniques can be applied to treat pain in the arms or legs caused by compressed spinal nerves. Historically, autogenous bone grafting, usually removed from the patient's iliac crest, has been used to increase fusion between vertebral levels.

[002] A protein that is osteogenic and commonly used to promote spine fusion is type 2 recombinant human bone protein (rhBMP-2). Small molecules have also been used to induce bone growth. Oxyesterols form a large family of oxygenated cholesterol derivatives that are present in the circulation and in human and animal tissues. It was found that oxyesterols are present in atherosclerotic lesions and play an important role in several physiological processes, such as cell differentiation, inflammation, apoptosis and steroid production. Some naturally occurring oxyesterols have robust osteogenic properties and can be used for bone growth. The most potent naturally occurring osteogenic oxyesterol, 20 (S) hydroxychloresterol, is both osteogenic and antiadipogenic when applied to multipotent mesenchymal cells capable of differentiating into osteoblasts and adipocytes.

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2/108 [003] One such oxy-cholesterol is Oxil33 or (3S, 5S, 6S, 8R, 9S, 10R, 13S,

14S, 17S) 17 - ((S) -2-hydroxyoctan-2-yl) -10,13-dimethylhexadecahydro-1Hcyclopenta [a] phenanthrene-3,6-diol, which exhibits the following structure:

[004] A variety of materials have been suggested for the treatment of bone defects. In addition to the traditional bone graft, several synthetic bone graft substitutes have been used or explored, including various matrix materials.

[005] In recent years, much attention has been paid to the supply of collagen-based materials, of human or animal origin. In particular, considerable attention has been directed to the development of preparations and materials based on animal tissues that are treated to provide compatibility, in order to avoid tissue rejection when used in humans. The main function of collagen in the dermal extracellular matrix is to act as a support in connective tissue. Predominantly, collagen is present in the form of type I collagen (80-85%) and type ΙΠ collagen (8-11%), both of which are fibrogens or rod-shaped collagens. The skin's tensile strength is due in large part to these collagen molecules that form in fibrils, with adjacent molecules cross-linking to further increase tensile strength. Collagen can also be used as a structural material to promote bone growth. Collagen has often been used as a carrier for compositions of injectable or preformed implants. The difficulty with compositions of injectable implants containing collagen, elastin and / or other biocompatible material is that there is a tendency for migration and resorption and this may mean that the implant is

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3/108 effective only for a limited time. Preformed implants are only suitable for specific procedures.

[006] Therefore, there is a need for improved malleable implants, which can be used to provide bone growth promoting agents. There is also a need for a collagen structure whose collagenase cannot break easily and can offer dimensional stability. In addition, there is a need to provide malleable implants that can incorporate an osteogenic agent, such as an oxy-cholesterol and to conveniently and quickly deliver oxy-cholesterol to a bone defect. In addition, there is also a need for a malleable implant with adhesive properties to attach to other medical implants, such as screws, nails, plates and intercorporeal devices comprising bone, allograft, autograft and / or synthetic materials.

SUMMARY [007] Implants containing oxy-cholesterol uniformly arranged in an acellular tissue matrix (ΑΊΜ) and methods for producing and using these implants are provided. The implants are configured to fit in a bone defect or close to a bone defect, to promote bone growth. In some embodiments, the implant is a gel, paste or moldable paste, which in some cases can be supplied by placing it in a syringe. In other modalities, the implant is lyophilized and, in some cases, placed in a syringe to supply a bone defect. After rehydration, the implant is hydrated and optionally placed in a syringe for easy application to a surgical site. In some embodiments, lyophilized implants have an acellular porcine collagen matrix, which may or may not be cross-linked, bone material and an osteogenic agent, for example, an oxy-cholesterol incorporated in them. In addition, moldable implants are provided which, in addition, can also include an expandable phase material or binder, for example carboxyl cellulose,

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4/108 alginate, pectin or chitosan. In one aspect, the present application is directed to an implantable osteogenic medical material comprising a moldable lyophilized implant that includes a combination of an acellular porcine collagen matrix, bone material and an active agent comprising an oxy-cholesterol.

[008] In some embodiments, a moldable implant is provided having an active agent that comprises the structure:

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R1 comprises an aliphatic or cyclic substituent having at least one carbon atom. In some embodiments, the active agent is a sterol comprising Oxil33.

[009] In certain embodiments, the implant's acellular tissue matrix (ATM) comprises solid or semi-solid particles of collagenous material that are derived from a natural tissue material, in which the collagenous material preserves the original fiber architecture and molecular ultrastructure of the natural tissue material from which it is derived, in which the collagenous material is substantially free of non-fibrous tissue proteins, glycoproteins, cellular elements, lipids or lipid residues, in which the collagenous material is not cytotoxic, in which the implant composition is it can be used as a component of a paste, gel or solution for injection, and in which the particles of collagenous material have a particle size in the range of approximately 50 microns to approximately 500 microns.

[0010] In other modalities, the acellular tissue matrix is a substantially non-antigenic, non-resorbable collagen fibrous tissue preparation, originating from human or animal tissue, which is suitable for homo- or heterotransplantation as a permanent repair of defects of the

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5/108 bones, the preparation of which retains the natural structure and original architecture of human or animal tissue, is substantially free of non-fibrous tissue proteins and glycoproteins, is substantially free of cellular elements, is substantially free of lipids and lipid residues and is not it is cytotoxic, in which the preparation is capable, when implanted, of being recolonized by the host cells (for example, osteoclasts, osteoblasts, etc.).

[0011] In several respects, the matrix of the acellular tissue is obtained from a human, swine, goat, bovine, ovine or equine source. In other respects, the matrix of the acellular tissue is porcine collagen, which in some cases is cross-linked porcine collagen.

[0012] In some embodiments, the acellular tissue matrix of the implant comprises from about 5% by weight to about 25% by weight of acellular porcine collagen and from about 0.01% by weight to about 90% in oxy-cholesterol weight. In other embodiments, in addition to oxy-cholesterol and an acellular tissue matrix, the implant comprises a fluid of about 25% by weight to about 50% by weight and bone material in an amount of about 25% by weight to about 75% by weight.

[0013] In certain modalities, the bone material of the implant includes demineralized bone matrix (DBM), mineralized bone matrix, demineralized bone fibers, demineralized bone chips. In other embodiments, the bone material may be inorganic ceramic comprising tricalcium phosphate, hydroxyapatite, biphasic calcium phosphate, porous calcium phosphate ceramic, tricalcium phosphate, hydroxyapatite or a combination thereof. In some respects, tricalcium phosphate and hydroxyapatite are in a ratio of about 70:30 to about 90:10 or in a ratio of about 85:15. In various embodiments, the particle size of the bone material is from about 125 pm to about 750 pm.

[0014] Fluids useful for implanting this disclosure include, without limitation, water, sodium chloride solution, dextrose, Ringer's solution with

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6/108

Lactate, blood, bone marrow aspirate, bone marrow fractions, phosphate buffered saline, DMSO, acetic acid, acetone, DME, DMF, MTBE, acetonitrile, butanol, butanone, t-butyl alcohol, ethanol, polyethylene glycol, methanol , chlorobenzene, chloroform, toluene, propanol, pentane, heptane, ethanol, Capriol-90 ™, diethyl ether or a combination thereof. The moldable implant can also include excipients, diluents, biodegradable polymers, intumescent agents, growth factors, antibiotics, solubilizers, stabilizers, thickening agents, antioxidants or binders. Useful binders or intumescent agents include carboxycylcellulose, alginate, chitosan, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, hydroxyethylcellulose, Carbopol, propylene glycol, polyvinyl alcohols, gelatin or a combination thereof.

[0015] In some embodiments, a lyophilized implant configured to fit into or near a bone defect site to promote bone growth is provided, the implant comprising a cross-linked acellular porcine collagen in an amount of about 0, 1 wt% to about 15 wt% of the implant and an oxy cholesterol in an amount of about 0.01 wt% to about 90 wt% of the implant, the oxy cholesterol having a concentration of about 100 mg / cc at about 1.0 g / cc.

[0016] In some embodiments, there is a method for preparing an implant, the method comprising mixing a fluid with an oxy-cholesterol, oxy-cholesterol in an amount of about 0.01% by weight to about 90% by weight based on weight total implant and in an acellular tissue matrix in an amount of about 0.1 wt% to about 20 wt% based on the total weight of the implant. In other embodiments, the method further comprises subjecting the implant mixed with the fluid for lyophilization to form a lyophilized implant, in which, in some cases, the acellular tissue matrix is acellular porcine collagen or an acellular porcine crosslinked collagen.

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7/108 [0017] In certain embodiments, the fluid used in this method is water, sodium chloride solution, dextrose, lactated Ringer's solution, phosphate buffered saline (PBS), blood, bone marrow aspirate, fractions of bone marrow or a combination of them in an amount of about 3/4 mL to about 10 mL. In other embodiments, the method further comprises adding an expandable phase material or binder in an amount of about 2% by weight to about 4% by weight based on the total weight of the implant. In some respects, the expandable phase or binder material is carboxyxyl cellulose, pectin, chitosan or a combination thereof.

[0018] In some embodiments, the method for preparing an implant also includes placing the implant in a mold before lyophilization. This mold can confer characteristics that increase the rehydration of the lyophilized implant, such as indentations or holes in random or defined ways. In other respects, the implant can be placed in a syringe, which in some ways can be placed in a vacuum-sealed bag. In other respects, the lyophilized implant can be placed in a vacuum sealed bag.

[0019] In some modalities, there is a method to treat a bone defect in a patient in need of it, the method comprising implanting in, or close to, a bone defect an implant comprising an oxycholesterol uniformly dispersed in an acellular tissue matrix. In some aspects, the implant used in the treatment method further comprises an expandable phase material or binder in an amount of about 2% by weight to about 4% by weight based on the total weight of the implant.

[0020] Additional features and advantages of various modalities will be presented in part in the description that follows, and in part will be evident from the description, or can be learned by practicing several

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8/108 modalities. The objectives and other advantages of various modalities will be realized and achieved by means of the elements and combinations particularly indicated in the description and in the attached claims.

BRIEF DESCRIPTION OF THE FIGURES [0021] In part, other aspects, characteristics, benefits and advantages of the modalities will be evident in relation to the description, attached claims and attached drawings below, in which

FIG. 1 illustrates an X-ray radiograph taken of a rat immediately after surgery, after having undergone a fusion of the two-level posterolateral lumbar spine where the implant was a malleable paste containing Permacol®, Oxil33 monohydrate, ceramics and carboxycylcellulose;

FIG. 2 illustrates an X-ray radiography taken of the same rat 4 weeks postoperatively after undergoing a fusion of the two-level posterolateral lumbar spine, where the implant was a malleable paste containing Permacol®, Oxil33 monohydrate , ceramics and carboxyl cellulose;

FIG. 3 illustrates an X-ray radiograph taken of the same rat 8 weeks postoperatively after undergoing a two-level posterolateral lumbar fusion in which the implant was a malleable paste containing Permacol®, Oxil33 monohydrate. , ceramics and carboxyl cellulose;

FIG. 4 illustrates a histology slide of a rat from the control group that was implanted with an implant material that did not contain any oxy-cholesterol including Oxil33 monohydrate;

FIG. 5 illustrates a histology slide (for example, H&E stain) from the same rat that underwent a two-level posterolateral lumbar spine fusion 8 weeks after the procedure where the implant

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9/108 was a malleable paste containing Permacol®, Oxil33 monohydrate, ceramic and carboxyl cellulose; and FIG. 6 illustrates a histology slide (for example, H&E staining) from the same rat that was subjected to fusion of the two-level posterolateral lumbar spine 8 weeks after the procedure where the implant was a malleable paste containing Permacol®, monohydrate of Oxil33, ceramics and carboxyl cellulose.

[0022] It will be understood that the numbers may not be scaled.

In addition, the relationship between objects in a figure may not be scaled, and may, in fact, have an inverse relationship to size. The figures intend to bring understanding and clarity to the structure of each object shown, and thus, some characteristics can be exaggerated to illustrate a specific characteristic of a structure.

DETAILED DESCRIPTION [0023] For the purposes of this specification and the appended claims, unless otherwise stated, all numbers expressing quantities of ingredients, percentages or proportions of materials, reaction conditions and other numerical values used in the specification and claims, should be understood as being modified in all examples by the term “about”. Consequently, unless otherwise indicated, the numerical parameters presented in the following specification and appended claims are approximations that may vary depending on the desired properties that are intended to be obtained through the present application. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter must at least be interpreted in light of the number of significant digits reported and by the application of common rounding techniques.

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10/108 [0024] Notwithstanding the numerical ranges and parameters that establish the broad scope of this application are approximations, the numerical values established in the specific examples are reported as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective test measurements. In addition, all of the tracks disclosed herein are to be understood as covering any and all of the sub-bands included therein. For example, a “1 to 10” range includes any and all sub-ranges between (and including) the minimum value of 1 and the maximum value of 10, that is, any and all sub-ranges with a minimum value equal to or greater than 1 and a maximum value less than or equal to 10, for example, 5.5 to 10.

Definitions [0025] It should be noted that, as used in this specification and in the appended claims, the singular forms “one”, “one” and “o, a” include plural referents unless expressly and unambiguously limited to one referent . Thus, for example, the reference to “an implant” includes one, two, three or more implants.

[0026] The term "bioactive agent" as used here is generally used to refer to any substance that alters a patient's physiology. The term "bioactive agent" can be used interchangeably here with the terms "therapeutic agent", "therapeutically effective amount" and "active pharmaceutical ingredient", "API" or "drug".

[0027] The term "biodegradable" includes compounds or components that will degrade over time by the action of enzymes, by the hydrolytic action and / or by other similar mechanisms in the human body. In many ways, “biodegradable” includes components that can break down or degrade within the body to non-toxic components as cells (eg bone cells) infiltrate the components and allow

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11/108 defect repair. By "bioerodible" is meant that compounds or components will erode or degrade over time due, at least in part, when they come into contact with substances found in the surrounding fluids of the tissue, or by cellular action. By "bioabsorbable" is meant that the compounds or components will be decomposed and absorbed in the human body, for example, by a cell or tissue. "Biocompatible" means that the compounds or components will not cause substantial tissue irritation or necrosis at the target tissue site and / or will not be carcinogenic.

[0028] The term "alkyl" as used here, refers to a saturated or unsaturated, branched, straight chain or cyclic hydrocarbon radical, derived from the removal of a hydrogen atom from a single carbon atom of an alkane, alkene or alkyne of origin. Typical alkyl groups include, but are not limited to, methyl; ethyl such as ethanyl, ethylene, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-in-1-yl, prop-2-in-1-yl, etc .; butyls like butan-

1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl-propan-2-yl, cyclobutan-1yl, but-l-en-l-yl, but-l-en- 2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-l-en-3-yl, cyclobut-1,3-dien-l-yl, but-l-in- l-yl, but-l-in-3-yl, but3-in-l-yl, etc .; and the like. When specific levels of saturation are desired, the nomenclature "alkenyl" and / or "alkynyl" is used, as defined below. In some embodiments, the alkyl groups are (C1-C40) alkyl. In some embodiments, the alkyl groups are (C1-C6) alkyl.

[0029] The term "alkanyl", as used here, refers to a straight, branched or cyclic, saturated alkyl radical, derived from the removal of a hydrogen atom from a single carbon atom of an origin alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethylinyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),

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12/108 cyclopropan-l-yl, etc .; butanyls such as butan-l-yl, butan-2-yl (secbutyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-l- ila, etc .; and the like. In some embodiments, the alkanyl groups are (C1-C40) alkanyl. In some embodiments, the alkanyl groups are (C1-C6) alkanyl.

[0030] The term "alkenyl", as used here, refers to a straight, branched or cyclic, unsaturated alkyl radical having at least one carbon-carbon double bond derived from the removal of a hydrogen atom from a single atom carbon of an alkene of origin. The radical may be in the useful or trans-conformation around the double bond (s). Typical alkenyl groups include, but are not limited to, ethylene; propenyls such as prop-l-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl, prop-2en-2-yl, cycloprop-l-en-l-yl ; cycloprop-2-en-1-yl; butenyls like but-l-en-

1-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-l-yl, but- 2-en-2-yl, buta-1,3-dien-l-yl, buta-1,3-dien-2-yl, cyclobut-l-en-l-yl, cyclobut-l-en-3- ila, cyclobut-1, 3-dien-1-yl, etc .; and the like. In some embodiments, the alkenyl group is (C2-C40) alkenyl. In some embodiments, the alkenyl group is (C2-C6) alkenyl.

[0031] The term "alkynyl" as used herein refers to an unsaturated branched or cyclic straight chain alkyl radical having at least one carbon-carbon triple bond derived by removing a hydrogen atom from a single carbon atom from one alkyne of origin. Typical alkynyl groups include, but are not limited to, ethynyl; propynyls, such as prop-1-in-1-yl, prop-2-in-1-yl, etc .; butynyls such as but-1-in-1-yl, but-3-in-1-yl, etc .; and the like. In some embodiments, the alkynyl group is (C2-C40) alkynyl. In some embodiments, the alkynyl group is (C2-C6) alkynyl.

[0032] The term "alkyldiyl" as used herein refers to a divalent saturated or unsaturated hydrocarbon radical, straight chain, branched,

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13/108 or cyclic derived from the removal of a hydrogen atom from each of the two different carbon atoms of an alkane, alkene or alkane of origin, or by the removal of two hydrogen atoms from a single carbon atom from an alkane, alkene or alkyne of origin. The two centers of monovalent radicals or each valence of the center of divalent radicals can form bonds with the same atoms or different atoms. Typical alkyldyls include, but are not limited to, methandylyls; ethyllines as ethan-

1.1- diyl, ethan-1,2-diyl, ethen-1, 1-diyl, eten-1,2-diyl; propyldiols such as propan-1, 1-diyl, propan-1,2-diyl, propan-2,2-diyl, propan-1,3-diyl, cyclopropan-1, l- di-ila, cyclopropan-1,2-di-yl, prop-l-en-1, l-di-ila, prop-l-en-

1.2-di-yl, prop-2-en-1,2,2-di-yl, prop-1-en-1,3-di-yl cycloprop-1-en-1,2-di-yl, cycloprop-2 -en-1, 2-diyl, cycloprop-2-en-1, 1-diyl, prop-1-in-1, 3-diyl, etc .; butyldiols such as butan-1, l-diyl, butan-1,2-diyl, butan-1,3-diyl, butanyl

1,4-diyl, butan-2,2-diyl, 2-methyl-propan-1, 1-diyl, 2-methyl-propan-1,2-diyl, cyclobutan-l, l- di-ila; cyclobutan-1,2-diyl, cyclobutan-1,3-diyl, but-l-en-

1,1-diyl, but-l-en-1,2,2-diyl, but-l-en-1,3-diyl, but-l-en-1,4-diyl, 2-methylprop-1 -en-1,1-di-yl, 2-methanylidene-propan-1,1-di-yl, buta-1,3-dien-1,1-diyl, buta-l, 3- dien-1,3-diyl, cyclobut-1-en-1,2-diyl, cyclobut-1-en-1,3-diyl, cyclobut-2-en-1,2-di- ila, cyclobut-1,3-dien-1,2-diyl, cyclobut-1,3-dien-1,3diyl, but-l-in-1,3-diyl, but-l- in-1, 4-diyl, buta-1, 3-di-in-1, 4-diyl, etc .; and the like. When specific levels of saturation are desired, the nomenclature alkanylhydric, alkenylhydric and / or alkynylhydric is used. In some embodiments, the alkyldiyl group is (C1-C40) alkyldiyl. In some embodiments, the alkyldiyl group is (C1-C6) alkyldiyl. Also contemplated are saturated acyclic alkanyldiyl radicals in which the radical centers are at the terminal carbons, for example, methandiyl (methane); ethan-1,2-diyl (ethane); propan-1,3-di-yl (propane); butan-1,4-diyl (butane); and the like (also referred to as alkylenes, defined below).

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14/108 [0033] The term "alkylene" as used here refers to a straight-chain alkyldiyl radical with two centers of terminal monovalent radicals derived from the removal of a hydrogen atom from each of the two terminal carbon atoms of alkane, alkene or alkaline of straight chain origin. Typical alkylene groups include, but are not limited to, methane; ethylenes such as ethane, ethylene, ethine; propylenes such as propane, prop [1] and ene, propa [1, 2] diene, prop [1], etc .; butylenes such as butane, but [l] ene, but [2] ene, buta [1,3] diene, but [l] ino, but [2] ino, but [1,3] diine, etc .; and the like. When specific levels of saturation are desired, the nomenclature alkane, alkene and / or alkaline is used. In some embodiments, the alkylene group is (C1-C40) alkylene. In some embodiments, the alkylene group is (C1-C6) alkylene.

[0034] The terms "heteroalkyl", "heteroalkanyl", "heteroalkenyl", "heteroalkynyl", "heteroalkylethyl" and "heteroalkylene", as used here, refer to alkyl, alkanyl, alkenyl, alkynyl, alkylhydryl radicals and alkylene, respectively, in which one or more of the carbon atoms are each independently substituted with the same or different heteroatomic groups. Typical heteroatomic groups that can be included in these radicals include, but are not limited to, —O—, —S—, —O — O—, —S — S—, —O — S—, NR ', = N — N =, —N = N—, —N (O) N—, _N = N — NR—, —PH—, —P (O) 2—, —O — P (O) 2—, —SH2—, - S (O) 2—, —SnH2— or the like, where each R 'is independently hydrogen, alkyl, alkanyl, alkenyl, alkynyl, aryl, arylaryl, arylalkyl, heteroaryl, heteroarylalkyl or heteroaryl-heteroaryl as defined herein.

[0035] The term "aryl" as used here refers to a monovalent aromatic hydrocarbon radical derived by removing a hydrogen atom from a single carbon atom from an original aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from aceantrylene, acenaftylene, acephenylelene, anthracene,

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15/108 azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexafene, hexylene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octafene, octalene, ovalene, penta-2,4-diene , pentacene, pentalene, pentafen, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinafthalene and the like. In some embodiments, the aryl group is (C5-C14) aryl or a (C5-C10) aryl group. Some aryls are phenyl and naphthyl.

[0036] The term "aryldiyl" as used here refers to a divalent aromatic hydrocarbon radical derived by removing a hydrogen atom from each of the two different carbon atoms in an original aromatic ring system or by removing two hydrogen atoms of a single carbon atom of an original aromatic ring system. The two centers of monovalent radicals or each valence of the divalent center can form bonds with the same or different atom (s). Typical aryldiyl groups include, but are not limited to, divalent radicals derived from aceanthylene, acenaphthene, acefenanthrene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorine, hexacene, hexafene, hexylene, asindacene, s-indacene, indane , indene, naphthalene, octacene, octafen, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentafene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaftilene and the like. In some embodiments, the group arildi-ila is (C5C14) arildi-ila or (C5-C10) arildi-ila. For example, some arildryl groups are divalent radicals derived from benzene and naphthalene, especially

1,4-diyl, naphtha-2,6-diyl and naphtha-2,7-diyl.

[0037] The term "arylene", as used here, refers to a divalent bridged radical having two adjacent monovalent radical centers derived from the removal of a hydrogen atom from each of the two adjacent carbon atoms in a ring system aromatic origin. The fixation of an arylene bridge radical, for example, benzene, to a

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16/108 original aromatic ring system, for example, benzene, results in a fused aromatic ring system, for example, naphthalene. The bridge is assumed to have the maximum number of non-cumulative double bonds consistent with its connection to the resulting fused ring system. In order to avoid double counting of carbon atoms, when an arylene substituent is formed by taking two adjacent substituents together in a structure that includes alternative substituents, the arylene bridge carbon atoms replace the bridged carbon atoms of the structure. As an example, consider the following structure:

R ² where R ¹ , when taken alone is hydrogen, or when taken together with R ² is (C5-C14) arylene; and R ² , when taken alone is hydrogen, or when taken together with R ¹ is (C5-C14) arylene.

[0038] When R ¹ and R ² are each hydrogen, the resulting compound is benzene. When R ¹ taken together with R ² is C6 arylene (benzene), the resulting compound is naphthalene. When R ¹ taken together with R ² is CIO arylene (naphthalene), the resulting compound is anthracene or phenanthrene. Typical arylene groups include, but are not limited to, aceanthylene, acenaphthene, acefenantilene, anthracene, azulene, benzene (benzo), chrysene, coronene, fluoranthene, fluorene, hexacene, hexafene, hexylene, as-indacene, s-indacene, indene , naphthalene (naphtho), octacene, octafen, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentafene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene or the like. When specific connectivity is desired, the bridged carbon atoms involved (from the arylene bridge) are denoted in parentheses, for example, [1, 2] benzene ([1, 2] benzo), [1, 2] naphthalene, [2, 3] naphthalene, etc. So in the

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17/108 example above, when R ¹ taken together with R ² is [2, 3] naphthalene, the resulting compound is anthracene. When R ¹ taken together with R ₂ is [1, 2] naphthalene, the resulting compound is phenanthrene. In one embodiment, the arylene group is (C5-C14) or (C5-C10).

[0039] The term "arylaryl", as used here, refers to a monovalent hydrocarbon radical derived from the removal of a hydrogen atom from a single carbon atom from a ring system in which two or more identical or aromatic ring systems non-identical are joined directly together by a single bond, where the number of these direct ring joints is one less than the number of original aromatic ring systems involved. Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaftyl, biphenyl-naphthyl and the like. When the number of carbon atoms comprising an arylaryl group is specified, the numbers refer to the carbon atoms comprising each aromatic ring of origin. For example, (C1-C14) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 14 carbons, for example, biphenyl, triphenyl, binaftyl, phenylnaphthyl, etc. In some cases, each aromatic ring system originating from an arylaryl group is independently an aromatic (C5-C14) or (C1-C10) aromatic. Some are arylaryl groups in which all aromatic ring systems of origin are identical, for example, biphenyl, triphenyl, binaftila, trinaftila, etc.

[0040] The term "biaryl", as used here, refers to an arylaryl radical with two identical aromatic systems of origin directly joined together by a simple bond. Typical biaryl groups include, but are not limited to, biphenyl, binaftyl, bianthracyl and the like. In some cases, aromatic ring systems are aromatic rings (C5-C14) or aromatic rings (C5-C10). In one embodiment, the biaryl group is biphenyl.

[0041] The term "arylalkyl", as used here, refers to an acyclic alkyl radical, in which one of the hydrogen atoms attached to an atom of

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18/108 carbon, typically a terminal carbon atom or spa, is replaced by an aryl radical. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,

2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and the like. When specific alkyl fractions are desired, the nomenclature arylalkanyl, arylalkenyl and / or arylalkynyl is used. In some embodiments, the arylalkyl group is (C6-C40) arylalkyl, for example, the alkanyl, alkenyl or alkynyl fraction of the (C1-C26) arylalkyl group and the aryl fraction is (C5-C14). In some embodiments, the arylalkyl group is (C6-C13), for example, the alkanyl, alkenyl or alkynyl fraction of the arylalkyl group is (C1-C3) and the aryl fraction is (C5-C10).

[0042] The term "heteroaryl" as used here refers to a monovalent heteroaromatic radical derived by the removal of a hydrogen atom from a single atom in a heteroaromatic ring system of origin. Typical heteroaryl groups include, but are not limited to, radicals derived from acridine, arsindol, carbazole, β-carboline, chroman, chromene, cinoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, quinoline, pyridine, quinoline, pyridine, quinoline tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene and the like. In some embodiments, the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 membered heteroaryl. Some heteroaryl radicals are those derived from heteroaromatic ring systems of origin in which any heteroatoms in the ring are nitrogens, such as imidazole, indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridine, pyridine, pyridine, pyridine, pyridine, pyridine, pyridine, pyridazine pyrrole, quinazoline, quinoline, etc.

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19/108 [0043] The term “heteroaryldiyl” refers to a divalent heteroaromatic radical derived by the removal of one hydrogen atom from each of two different atoms from a heteroaromatic ring system of origin or by the removal of two atoms hydrogen from a single atom of a heteroaromatic ring system. The two centers of monovalent radicals or each valence of the single divalent center can form bonds with the same or different atom (s). Typical heteroaryldiyl groups include, but are not limited to, divalent radicals derived from acridine, arsindol, carbazole, β-carboline, chroman, chromene, cinoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isoindol, isoindol , isoindoline. isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazine, pyridazine, pyridine, pyrimidine, quinoline, pyrrolidine, quinoline, pyridine, quinoline , thiadiazole, thiazole, thiophene, triazole, xanthene and the like. In some embodiments, the heteroarylldiyl group is a 5-14 membered heteroaryldiyl or a 5-10 membered heteroaryldiyl. Some heteroaryl-diyl groups are divalent radicals derived from heteroaromatic ring systems of origin in which any ring heteroatoms are nitrogens, such as imidazole, indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine, pyrazine, pyrazine, pyrazine , pyridine, pyrrole, quinazoline, quinoline, etc.

[0044] The term "heteroarylene" as used here refers to a divalent bridged radical having two centers of adjacent monovalent radicals derived by the removal of one hydrogen atom from each of two adjacent atoms from an originating heteroaromatic ring system. The attachment of a heteroarylene bridge radical, for example, pyridine, to an original aromatic ring system, for example, benzene, results in a fused heteroaromatic ring system, for example, quinoline. The bridge is assumed to have the maximum number of non-cumulative double bonds

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20/108 consistent with its connection to the resulting fused ring system. In order to avoid double counting ring atoms, when a heteroarylene substituent is formed by taking two adjacent substituents together in a structure that includes alternative substituents, the heteroarylene ring atoms replace the bridge's ring atoms in the structure. As an example, consider the following structure:

R ² where R ¹ , when taken alone is hydrogen, or when taken together with R ² is 5-14 membered heteroarylene; and R ² , when taken alone is hydrogen, or when taken together with R ¹ is 5-14 membered heteroarylene;

[0045] When R ¹ and R ² are each hydrogen, the resulting compound is benzene. When R ¹ taken together with R ² is a 6-membered pyridine heteroarylene, the resulting compound is isoquinoline, quinoline or quinolizine. When R ¹ taken together with R ² is a 10-membered heteroarylene (for example, isoquinoline), the resulting compound is, for example, acridine or phenanthridine. Typical heteroarylene groups include, but are not limited to, acridine, carbazole, β-carboline, chromene, cinolin, furan, imidazole, indazolno, indolene, indolizine, isobenzofuran, isochromene, isoindolene, isoquinoline, isothiazolno, oxoxazole, isoxazole , perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazoline, pyridazine, pyridine, pyrimidine, pyrrolene, pyrrolizine, quinazoline, quinoline, quinoline, quinoxaline, thiazolin, thiazolin, thiazoline , or the like. When a specific connectivity is desired, the bridged atoms involved (from the heteroarylene bridge) are indicated in parentheses, for example, [1,2,2] pyridine, [2,3] pyridine, [3,4] pyridine, etc. So, in the example above, when R ¹ taken together

Petition 870180049765, of 11/06/2018, p. 29/129 / 108 with R ² is [1,2] pyridine, the resulting compound is quinolizine. When R ¹ taken together with R ² is [2,3] pyridine, the resulting compound is quinoline. When R ¹ taken together with R ² is pyridine [3,4], the resulting compound is isoquinoline. In some embodiments, the heteroarylene group is 5-14 membered heteroarylene or 5-10 membered heteroarylene. Some heteroarylene radicals are those derived from heteroaromatic ring systems of origin in which any heteroatoms in the ring are nitrogens, such as imidazole, indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridazine, pyrazine, pyridazine pyrrole, quinazoline, quinoline, etc.

[0046] The term "heteroaryl-heteroaryl" as used here refers to a monovalent heteroaromatic radical derived by removing a hydrogen atom from a single atom in a ring system in which two or more heteroaromatic ring systems of origin identical or non-identical are joined directly together by a single bond, in which the number of such direct ring junctions is one less than the number of heteroaromatic ring systems involved. Typical heteroarilheteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc. When the number of ring atoms is specified, the numbers refer to the number of atoms comprising each heteroaromatic ring system of origin. For example, 5-14 membered heteroaryl heteroaryl is a heteroaryl-heteroaryl group in which each heteroaromatic ring system of origin comprises 5 to 14 atoms, for example, bipyridyl, tripyridyl, etc. In some embodiments, each heteroaromatic ring system of origin is independently a 5-14 membered heteroaromatic, in other embodiments, a 5-10 membered heteroaromatic. There are also heteroaryl-heteroaryl groups in which all heteroaromatic ring systems of origin are identical. Some heteroaryl-heteroaryl radicals are those in which each heteroaryl group is derived from

Petition 870180049765, of 11/06/2018, p. 30/129 / 108 heteroaromatic ring systems of origin in which any heteroatoms in the ring are nitrogens, such as imidazole, indole, indazole, isoindole, naphthyridine, pteridine, isoquinoline, phthalazine, purine, pyrazole, pyrazine, pyridine, pyridine, pyrrole, quinazoline, quinoline, etc.

[0047] The term "bi-heteroaryl", as used here, refers to a heteroaryl-heteroaryl radical having two identical heteroaromatic ring systems, joined directly together by a single bond. Typical bi-heteroaryl groups include, but are not limited to, bipyridyl, bipurinyl, biquinolinyl and the like. In some embodiments, heteroaromatic ring systems are 5-14 membered heteroaromatic rings or 5-10 membered heteroaromatic rings. Some bi-heteroaryl radicals are those in which heteroaryl groups are derived from a heteroaromatic ring system of origin in which any ring heteroatoms are nitrogens, such as bi-imidazolyl, bi-indolyl, bi-indazolyl, bi-isoindolyl, binafatiridinyl , biperidinyl, bi-isoquinolinyl, bifiralazinyl, bipurinyl, bipyrazolyl, bipyrazinyl, bipyridazinyl, bipyridinyl, bipyrrolyl, bikiniinazolinyl, bikiniinolinyl, etc.

[0048] The term "heteroarylalkyl" as used here refers to an acyclic alkyl radical in which one of the hydrogen atoms attached to a carbon atom, typically a terminal carbon atom or sp2, is replaced with a heteroaryl radical. When specific alkyl fractions are desired, the nomenclature heteroarylalkanyl, heteroarylalkenyl and / or heteroalkynyl is used. In some embodiments, the heteroarylalkyl group is a 6-20 membered heteroarylalkyl, for example, the alkanyl, alkenyl or alkynyl fraction of the heteroarylalkyl has 1-6 members and the heteroaryl fraction is a 5-14 membered heteroaryl. In some embodiments, heteroarylalkyl is a 6-13 membered heteroarylalkyl, for example, the alkanyl, alkenyl or alkynyl fraction has 1-3 members and the heteroaryl fraction is a 5-10 membered heteroaryl.

Petition 870180049765, of 11/06/2018, p. 31/129 / 108 [0049] The term "substituted" as used here refers to a radical in which one or more hydrogen atoms are each, independently, substituted with the same or different (s) ) substituent (s). Typical substituents include, but are not limited to, —X, —R, —O-, = 0, —OR, —O — OR, —SR, —S-, = S, —NRR, = NR, perhalo ( C1-C6) alkyl, —CX3, —CF3, —CN, —OCN, —SCN, —NCO, —NCS, —NO, - NO2, = N2, —N3, —S (O) 2O-, —S ( O) 2OH, —S (O) 2R, —C (O) R, —C (O) X, —C (S) R, —C (S) X, —C (O) OR, —C (O ) O-, —C (S) OR, —C (O) SR, - C (S) SR, —C (O) NRR, —C (S) NRR and —C (NR) NRR, where each X is independently a halogen (e.g., -F or -Cl) and each R is independently hydrogen, alkyl, alkanyl, alkenyl, alkanyl, aryl, arylalkyl, arylaryl, heteroaryl, heteroarylalkyl or heteroarylheteroaryl, as defined herein. The actual substituent that replaces any particular group will depend on the identity of the group being replaced.

[0050] The term "solvate", as used here, refers to an aggregate comprising one or more molecules of a compound of the disclosure with one or more molecules of solvent. The solvent can be water, in which case the solvate can be a hydrate. Alternatively, the solvent can be an organic solvent. Thus, the compounds of the present disclosure can exist as a hydrate, including a monohydrate, dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate or a combination thereof, as well as the corresponding solvated forms . The compounds of the disclosure can be true solvates, while in other cases, the compounds of the disclosure can only retain accidental water or be a mixture of water plus some adventitious solvent.

[0051] The term "oxy-cholesterol" as used here is intended to encompass one or more forms of oxidized cholesterol. The oxyesterols described here are either independently or collectively active for bone growth

Petition 870180049765, of 11/06/2018, p. 32/129 / 108 in a patient, as described in WO 2013169399 A1, which is incorporated herein by reference in its entirety.

[0052] Oxy-cholesterol may be in the form of a pharmaceutically acceptable salt. Some examples of potentially pharmaceutically acceptable salts include the salt-forming acids and bases that do not substantially increase the toxicity of a compound, such as alkali metal salts such as magnesium, potassium and ammonium, mineral acid salts such as hydrochloride, hydroiodic acid, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric, as well as salts of organic acids such as tartaric, acetic, citric, malic, benzoic, glycolic, gluconic, gulonic, succinic, arylsulfonic acids, for example, p-toluenesulfonic acids, or the like .

[0053] Pharmaceutically acceptable salts of oxy-cholesterol include salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases, inorganic or organic acids and fatty acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganous, potassium, sodium, zinc salts and the like. Salts derived from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins such as arginine, betaine, caffeine, choline, N, N'dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, Netylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, procamine, pyrine, polyamine, procamine, pyrine, polyamine, procamine, pyrine, polyamine, proclin, pyrine, polyamine, pyramine, proclin, pyrine purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like. When the compound of the present patent application is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids,

Petition 870180049765, of 11/06/2018, p. 33/129 / 108 including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, malonic, mucinic, pantonic, nitric phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonic, trifluoroacetic acid and the like. Fatty acid salts can also be used, for example, fatty acid salts that contain more than 2 carbon atoms, more than 8 carbon atoms or more than 16 carbon atoms, such as butyric, caproic, caprylic, capric, lauric , mystical, palmitic, stearic, arachian or similar.

[0054] In some embodiments, in order to reduce the solubility of oxy-cholesterol to assist in obtaining a controlled release deposit effect, oxy-cholesterol used as a free base or used in a salt that has a relatively lower solubility. For example, the present patent application can use an insoluble salt, such as a fatty acid salt. Representative fatty acid salts include oleic acid, linoleic acid or fatty acid salts with a solubility of between 8 to 20 carbons, such as, for example, palmeate or stearate.

[0055] The terms "bioactive" composition or "pharmaceutical" composition as used here, can be used interchangeably. Both terms refer to compositions that can be administered to an individual. Bioactive or pharmaceutical compositions are sometimes referred to here as "pharmaceutical compositions" or "bioactive compositions" in the disclosure. Sometimes the phrase "administration of Oxil33" is used here in the context of administering this compound to an individual (for example, contacting the individual with the compound, injecting the compound, administering the compound into an implant, etc.). It is to be understood that the compound for such use can generally be in the form of a pharmaceutical composition or bioactive composition comprising oxy-cholesterol (for example, Oxil33).

Petition 870180049765, of 11/06/2018, p. 34/129 / 108 [0056] A "therapeutically effective amount" or "effective amount" is such that, when administered, oxy-cholesterol (eg, Oxil33) results in alteration of biological activity, such as, for example, growth enhancement bone, etc. The dosage administered to a patient can be as single or multiple doses depending on a variety of factors, including the pharmacokinetic properties of the drug administered, the route of administration, conditions and characteristics of the patient (sex, age, body weight, health, size, etc.) and extent of symptoms, concomitant treatments, frequency of treatment and the desired effect. In some embodiments, the formulation is designed for immediate release. In other modalities, the formulation is designed for sustained release. In other embodiments, the formulation comprises one or more immediate release surfaces and one or more sustained release surfaces.

[0057] A "deposit" includes, but is not limited to, capsules, microspheres, microparticles, microcapsules, microfiber particles, nanospheres, nanoparticles, coatings, matrices, wafers, pills, lozenges, emulsions, liposomes, micelles, gels or others pharmaceutical compositions or a combination thereof. Suitable materials for the deposit are ideally biodegradable and / or bioabsorbable pharmaceutically acceptable materials that are FDA approved or GRAS materials. These materials can be polymeric or non-polymeric, as well as synthetic or naturally occurring, or a combination thereof. In some embodiments, the matrix can be a biodegradable deposit.

[0058] The term "implantable", as used here, refers to a biocompatible device (eg, implant) that retains the potential for successful placement in a mammal. The term “implantable device” and similar import expressions, as used herein, refer to an implantable object through surgery, injection or

Petition 870180049765, of 11/06/2018, p. 35/129 / 108 other suitable means whose main function is achieved through their physical presence or mechanical properties.

[0059] The “localized” application includes the application where one or more drugs are deposited within a tissue, for example, a bone cavity, or nearby (about 0.1 cm, or in many cases, about 10 cm, for example) of the same. For example, oxy-cholesterol administered locally from the implant can be, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or 99.999% less than the oral or injectable dose.

[0060] The term "mammal" refers to organisms of the taxonomic class "mamalia", including, but not limited to humans, other primates such as monkeys, chimpanzees, apes, orangutans, rats, mice, rabbits, cats, dogs , pigs, cows, horses, etc.

[0061] The term “particle” refers to pieces of a substance of all shapes, sizes, thicknesses and configurations, such as fibers, threads, narrow strips, thin sheets, clips, fragments, etc., which have regular geometries, irregular or random. It should be understood that some variation in dimension will occur in the production of particles and particles demonstrating such variability in dimensions are within the scope of the present application. For example, mineral particles (for example, ceramic) can be from about 0.5 mm to about 1.5 mm. In some embodiments, the mineral particles can be from about 0.2 mm to about 0.5 mm.

[0062] In some embodiments, the medical device comprises a matrix. The "matrix" of the present application is used as a support for repair, regeneration and / or augmentation of bone and / or cartilage. Typically, the matrix provides a 3-D matrix of interconnecting pores, which act as a support for cell migration. The matrix morphology guides cell migration and cells are able to migrate into or over the matrix, respectively. The cells are then able to proliferate and

Petition 870180049765, of 11/06/2018, p. 36/129 / 108 synthesize new tissues and form bones and / or cartilage. In some embodiments, the matrix is resorbable and comprises particles of collagen and ceramic and an oxy-cholesterol uniformly distributed throughout the matrix.

[0063] In some embodiments, the matrix can be malleable, cohesive, flowable and / or can be shaped in any shape. The term “malleable” includes the fact that the matrix is capable of being converted from a first form to a second form by applying pressure.

[0064] The term "cohesive", as used here, means that the matrix tends to remain a singular mass connected by movement, including the display of the ability to stretch substantially without breaking with stretching. An example of a cohesive matrix includes, for example, a mass.

[0065] The term “moldable” includes that the matrix can be molded by hand or machine or injected into the target tissue site (for example, defect, fracture or bone void) in a wide variety of configurations. In some embodiments, the matrix may be formed of sheets, blocks, rings, struts, plates, discs, cones, pins, screws, tubes, teeth, bones, bone portion, wedges, cylinders, threaded cylinders or the like, as well as more complex geometric configurations.

[0066] Oxy-cholesterol can be "osteogenic", where it can intensify or accelerate the growth of new bone tissue.

[0067] The term "acellular tissue matrix" ("ATM") refers to a tissue-derived structure that is made from any of a wide range of collagen-containing tissues, removing all, or substantially all, cells viable and, in many cases, all detectable dead cells, subcellular components and / or debris generated by dead or dying cells. As used here, an “acellular matrix” is a matrix that: (a) is made from any of a wide range of tissues based on

Petition 870180049765, of 11/06/2018, p. 37/129 / 108 collagen; (b) is acellular; and (c) it retains the biological and structural functions possessed by the native tissue or organ from which it was made.

[0068] The term "decellularize" refers to a process that eliminates or sufficiently reduces native cells and cellular material in a tissue such that, when this tissue is implanted, it does not invoke an adverse immune response. The term "acellular" is intended to refer to tissue that is sufficiently reduced in cells and cellular material so as not to invoke an adverse immune response.

[0069] As used here, “freeze drying” or “freeze drying” involves drying a material in a frozen state at very low pressure (for example, in a high vacuum), so that ice, or another frozen solvent, it sublimates quickly without melting. Methods and apparatus for lyophilization are well known in the art (see, for example, “FreezeDrying / Lyophilization Of Pharmaceutical and Biological Productsf Third Edition, Louis Rey and Joan C. May, Informa Healthcare, 2010). The lyophilization step can be carried out by any suitable method and using any suitable apparatus.

[0070] The headings in the sections below should not be restricted and may be interchanged with other headings in the section.

Oxyesterols [0071] Implants containing oxycholesterol and methods for producing and using these implants are provided. Upon rehydration, implants can be formed to easily fit within a bone defect. In some embodiments, the implants have an acellular porcine collagen that may or may not be cross-linked, a bone material and an osteogenic agent, for example, an oxy-cholesterol incorporated within them. In addition, moldable implants are provided which, in addition, may also include an expandable phase material or binder, for example, carboxyethyl cellulose, alginate, pectin or chitosan. In one respect, the

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The present application is directed to an implantable osteogenic medical material comprising a moldable implant that includes a combination of an acellular porcine collagen, bone material and an active agent comprising an oxy-cholesterol.

[0072] In some modalities, the malleable implant can be a robust implant that contains allograft materials or minerals that can still bond and be cohesive. In some embodiments, there is a malleable implant configured to fit, or approach, a bone defect to promote bone growth, the malleable implant comprising an acellular porcine collagen in an amount of about 0.1% by weight at about 20% by weight of the implant, bone material in an amount of about 0.1% by weight at about 95% by weight of the implant, and an oxy-cholesterol, the implant configured to become moldable when moistened with a fluid.

[0073] Oxyesterols are a family of molecules that consist of oxygenated derivatives of cholesterol. Oxyesterols are involved in many biological processes, and have been found to have osteogenic properties. For example, a naturally occurring oxy-cholesterol, 20 (S) hydroxycholesterol, has osteogenic and antiadipogenic properties. Such oxyesterols can be useful in healing bone fractures, long bone fusion procedures, spine fusion procedures, intercorporeal spine fusion procedures, posterolateral spine fusion procedures, and cervical discectomy and fusion procedures, procedures dental and cranial / maxillofacial procedures.

[0074] Oxyesterols also play a role in various physiological processes, such as cell differentiation, inflammation, apoptosis, and steroid production. Oxyesterols are products of cholesterol oxidation and are formed in vivo by a variety of cell types, including osteoblasts (Schroepfer. Phyiol Rev 80: 361-554, 2000; Bjorkhem

Petition 870180049765, of 11/06/2018, p. 12/39

10/31 and Dicsfalusy. Arterioscler Tromb Vase Biol 22: 734-742, 2002). Certain oxyesterols, such as 20 (S) -hydroxycholesterol, as well as 22 (S) - or 22 (R) hydroxycholesterol, induce osteogenic differentiation in multipotent mesenchymal cells, such as M2-10B4 bone marrow stromal cells ( M2), and embryonic fibroblasts C3H10T1 / 2 (Kha et al. J Bone Miner Res 19: 830-840, 2004). Oxyesterols can induce osteogenesis and inhibit adipogenic differentiation of mesenchymal stem cells, by activating the hedgehog signaling pathway, which in turn regulates the main switches that control osteogenic and adipogenic differentiation, namely Runx2 and PPARy, respectively ( Richardson et al. J Cell Biochem 100: 1131-1145, 2007; Dwyer et al. J Biol Chem 282: 8959-8968, 2007; Kim et al., J Bone Miner Res 22: 1711-1719, 2007). Some oxyesterols also provide therapeutic uses for treating bone defects or disorders such as osteoporosis.

[0075] The implants described in this document can be useful in the creation of new therapeutic implants and matrices that include an oxy-cholesterol to induce local bone formation and treat bone defects. Oxy-cholesterol is retained in the TMJ and released over time, while the TMJ allows the influx of bone cells to grow and fill the defect. In some embodiments, these applications are based on the ability of these oxy-cholesterol compounds to induce the hedgehog signaling pathway. In some modalities, the implant causes the mesenchymal stem cells to show the induced expression of osteoblast differentiation markers. The implants and matrices described herein can be used for a variety of therapeutic uses including, but not limited to, inducing local bone formation and treating bone defects. In some embodiments, implants containing oxycholesterol as described herein induce a biological response when the implant contacts a human or animal cell. In some embodiments, the cell can be a cell

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32/108 mesenchymal trunk or a bone marrow stromal cell. In some modalities, the biological response includes stimulating osteoblastic differentiation, inhibiting adipocyte differentiation or stimulating scar formation. In some embodiments, the implant is configured as an implant to release oxy-cholesterol to induce a biological response at, or close to, a surgical site or a bone defect site.

[0076] Oxyesterols can be used to induce systemic bone formation for the treatment of bone defects, such as osteoporosis, to induce bone formation at the site for the treatment of conditions such as jointless fractures, or other bone disorders, such as jaw bone defects in dental applications / implants and to induce spine fusion. In some embodiments, the implant may include an oxy-cholesterol alone or in combination with one or more bone morphogenetic proteins or osteogenic agents. In some embodiments, more than one oxy-cholesterol is present in the implant. In some embodiments, implants include Oxil33 and / or Oxil53.

[0077] In some modalities, the implant or the TMJ include oxyesterols that aid in osteogenesis. In some embodiments, the implant or the ATM include Oxi34, Oxi49 and / or Oxil33. In some embodiments, the implant or the ATM includes an oxy-cholesterol comprising the structure:

or a pharmaceutically acceptable salt, hydrate or solvate thereof, wherein R ¹ comprises an aliphatic or cyclic substituent having at least one carbon atom.

[0078] In some embodiments, Ri comprises an alkyl, heteroalkyl, an alkanyl, a heteroalkanyl, an alkenyl, a heteroalkenyl, an alkynyl, a heteroalkynyl, an alkyldyl,

Petition 870180049765, of 11/06/2018, p. 41/129 / 108 heteroalkylethyl, an alkylene, a heteroalkylene, an aryl, an aryldiyl, an aridene, an arylaryl, a biaryl, an arylalkyl, a heteroaryl, a heteroarylhyldyl, a heteroarylene, a heteroaryl-heteroaryl , a biheteroaryl, a heteroarylalkyl or combinations thereof. In some embodiments, the R1 substituent comprises an (C1-C20) alkyl or heteroalkyl, one (C2-C20) aryl or heteroaryl, one (C6-C26) arylalkyl or heteroaryl-heteroalkyl and one (C5-C20) arylalkyl or heteroaryl- heteroalkyl, a (C4-C10) alkyldiyl or heteroalkyldiyl, or a (C4-C10) alkylene or heteroalkylene. The R1 substituent may be cyclic or acyclic, branched or unbranched, substituted or unsubstituted, aromatic, saturated or unsaturated, or combinations thereof. In some embodiments, the substituent Ri is an aliphatic group. In some embodiments, the substituent Ri is a cyclic group. In some embodiments, the substituent Ri is a hexyl group.

[0079] The present disclosure includes an implant, including osteogenic oxy-cholesterol (eg, Oxil33) uniformly disposed (eg, dispersed) in an ATM, and oxy-cholesterol has the ability to promote osteogenic differentiation in vitro. Oxil33 is a particularly effective osteogenic agent. In several applications, Oxil33 is useful in the treatment of conditions that would benefit from localized stimulation of bone formation, such as, for example, spinal fusion, fracture repair, tissue applications / bone regeneration, increased bone density in the mandible for implants dental, osteoporosis or the like. A particular advantage of Oxil33 is that it provides greater ease of synthesis and a better melting time when compared to other osteogenic oxyesterols. Oxil33 is a small molecule that can serve as an anabolic therapeutic agent for bone growth, as well as a useful agent for treating a variety of other conditions.

Petition 870180049765, of 11/06/2018, p. 42/129 / 108 [0080] The compositions and methods for the preparation of Oxil33 were described in International Application PCT / 2015/064526, filed on December 8, 2015, the content of which is incorporated herein by reference in its entirety.

[0081] One aspect of the disclosure of the patent application is an implant or an ATM including Oxil33, having the formula:

or a pharmaceutically acceptable salt, solvate or hydrate thereof. Oxil33 can be used as a pharmaceutical or bioactive composition comprising Oxil33 or a pharmaceutically acceptable salt, solvate or hydrate thereof and a pharmaceutically acceptable carrier. Oxil33 has the designation IUPAC (35,55,65,8 / ?, 95,10 / ?, 135,145,175) - 17 - ((5) -2-hydroxyoctan-2-yl) -10,13dimethyl-hexadecahydro-1 H- cyclopentin [a] phenanthrene-3,6-diol.

[0082] Another aspect of the disclosure is a method for the induction (stimulation, intensification) of a response mediated by the hedgehog (Hh) pathway, in a cell or tissue, comprising contacting the cell or tissue with a therapeutically effective amount of Oxil33. The cell or tissue can be in vitro or in an individual, such as a mammal. The response mediated by the hedgehog (Hh) pathway involves the stimulation of osteoblastic differentiation, osteomorphogenesis and / or osteoproliferation; stimulation of hair growth and / or cartilage formation; the stimulation of neovasculogenesis, for example, angiogenesis, thereby intensifying the blood supply to ischemic tissues; or it is the inhibition of adipocyte differentiation, adipocyte morphogenesis and / or adipocyte proliferation; or stimulation of progenitor cells to suffer

Petition 870180049765, of 11/06/2018, p. 43/129 / 108 neurogenesis. The Hh-mediated response may comprise the regeneration of any of a variety of tissue types, for use in regenerative medicine. Another aspect of the disclosure is a method for treating an individual with a bone disorder, osteopenia, osteoporosis or a bone fracture, comprising administering to the individual an effective amount of a bioactive composition or pharmaceutical composition comprising Oxil33. The individual can administer the bioactive composition or the pharmaceutical composition at a therapeutically effective dose, in an effective dosage form at a selected interval, for example, to increase bone mass, improve osteoporosis symptoms, reduce, eliminate, prevent or treat atherosclerotic lesions, or the like. The individual can administer the bioactive composition or the pharmaceutical composition in a therapeutically effective dose, in an effective dosage form at a selected interval to improve the symptoms of osteoporosis. In some embodiments, a composition comprising Oxil33 can include mesenchymal stem cells to induce osteoblastic differentiation of cells in a specific surgical area.

[0083] In several respects, Oxil33 can be administered to a cell, tissue or organ by local administration. For example, Oxil33 can be applied locally with a cream or the like, or it can be injected or introduced directly into a cell, tissue or organ, or it can be introduced with a suitable medical device, such as an implant as discussed here.

[0084] In some embodiments, the dosage of Oxil33 is approximately 10 pg / day to about 80 g / day. In some embodiments, the dosage of Oxil33 is about 1.0 g / day, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0 , 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11 , 5, 12.0, 12.5 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24, 0, 24.5, 25.0 25.5 26.0 26.5

Petition 870180049765, of 11/06/2018, p. 12/44/ 108

27.0 27.5 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39, 5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5 50.0, 50.5, 51.0, 51.5, 52.0 , 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58 .5, 59.0, 59.5 to about 60.0 grams / day. Additional dosages of Oxil33 include approximately 2.4 ng / day to approximately 50 mg / day; approximately 50 ng / day at

about 2.5 mg / day; about 250 ng / day The about 250 mcg / day; about 250 ng / day The about 50 mcg / day; about 250 ng / day The about 25 mcg / day; about 250 ng / day The about 1 mcg / day; about 300 ng / day The

approximately 750 ng / day or approximately 0.50 mcg / day at 500 ng / day. In various embodiments, the dose can be from about 0.01 to approximately 10 mcg / day or approximately 1 ng / day to about 120 mcg / day. In some modalities, the dosage of Oxil33 is in greater quantities. For example, in some embodiments, the dosage of Oxil33 is 0.01 mg / day to 5 g / day.

[0085] An acellular tissue matrix (ATM) can comprise oxy-cholesterol (for example, Oxil33) homogeneously disposed throughout the region or in discrete regions or discrete layers of the ATM. Oxy-cholesterol can be loaded into the ATM and can comprise from about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0 , 1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5,

Petition 870180049765, of 11/06/2018, p. 12/45/ 108

40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0,

47.5, 48.0, 48.5, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0,

54.5, 55.0, 55.5, 56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5 to about 60% w / v, w / w and / or v / v of the total weight of the ATM.

[0086] In some embodiments, a high concentration of oxy-cholesterol can be carried in the ATM and comprises from about 20% by weight to about 99% by weight of the ATM. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 20% by weight to about 90% by weight, 35% by weight to about 80% by weight or about 50% by weight to about 90% by weight of the ATM. In some embodiments, the oxy-cholesterol can be loaded into the ATM in an amount of about 20% by weight to about 30% by weight, about 30% by weight to about 40% by weight, about 40% by weight to about 50% by weight, about 50% by weight to about 60% by weight, about 60% by weight to about 70% by weight, about 70% by weight to about 80% by weight, about from 80% by weight to about 90% by weight or about 90% by weight to about 99% by weight. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, a about 99% by weight of the ATM.

[0087] Oxy-cholesterol can be loaded in the ATM and can comprise from about 0.01, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0, 45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,

6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19, 0, 19.5, 20.0, 20.5, 21.0, 21.5, 22.0, 22.5, 23.0, 23.5, 24.0, 24.5, 25.0, 25.5, 26.0, 26.5, 27.0, 27.5, 28.0, 28.5, 29.0, 29.5, 30.0, 30.5, 31.0, 31, 5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36.0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44, 0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48.5, 49.0, 49.5, 50.0, 50.5, 51.0, 51.5, 52.0, 52.5, 53.0, 53.5, 54.0, 54.5, 55.0, 55.5,

Petition 870180049765, of 11/06/2018, p. 46/129

10/38

56.0, 56.5, 57.0, 57.5, 58.0, 58.5, 59.0, 59.5 and about 60 mg / cc of ATM. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 400 mg / cc. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220 , 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470 , 480, 490, at about 500 mg / cc. In some embodiments, 400 mg / cc can be loaded at the ATM.

[0088] In some modalities, oxy-cholesterol can be loaded into the

ATM in highly concentrated quantities. For example, in some embodiments, oxy-cholesterol is loaded into the ATM in an amount of at least 500 mg / cc. In some embodiments, oxy-cholesterol is added to the ATM in an amount of about 1 mg / cc to about 1 g / cc, from about 100 mg / cc to about 1 g / cc, from about 500 mg / cc at about 900 mg / cc, or from about 600 mg / cc to about 800 mg / cc. In other embodiments, oxy-cholesterol is added to the ATM in an amount of about 500 mg / cc to about 600 mg / cc, from about 600 mg / cc to about 700 mg / cc, from about 700 mg / cc at about 800 mg / cc, from about 800 mg / cc to about 900 mg / cc or from about 900 mg / cc to about 1 g / cc.

[0089] In addition to the Oxil33 compound, other modalities of disclosure encompass any and all individual stereoisomers in any of the stereocenters present in Oxil33, including diastereoisomers, racemates, enantiomers, and other isomers of the compound. In the disclosure modalities, Oxil33 can include all polymorphs, solvates or hydrates of the compound, such as hydrates and those formed with organic solvents. In various embodiments, Oxil33 includes polymorph of Form A, polymorph of Form B, polymorph of Form C, polymorph of Form D, polymorph of Form E, polymorph of Form F, polymorph of Form G, polymorph of Form H, polymorph of Form I or a mixture of them

Petition 870180049765, of 11/06/2018, p. 47/129 / 108 as described in US 15 / 082,695 and 15 / 374,610 incorporated herein by reference as if they were presented in full.

[0090] In some embodiments, the oxy-cholesterol that can be uniformly distributed in the acellular tissue matrix can be an Oxil33 polymorph which comprises, consists essentially of, or consists of Form A polymorph, and / or Form B polymorph. modalities, the polymorph of Oxil33 comprises, essentially consists of, or consists of Form A which produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 16.4, 17.91 and 20.94 ± 0 , 2 degrees 2Θ. In some embodiments, the Oxil33 polymorph comprises, consists essentially of, or consists of Form A which produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 6.1, 12.3 and 18.6 ± 0.2 degrees 2Θ instead of or in addition to the X-ray diffraction pattern of the above for Form A.

[0091] In some embodiments, Oxil33 polymorph which can be uniformly distributed in the matrix of the acellular tissue comprises, consists essentially of, or consists of Form B which produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 13.3, 16.1 and 18.82 ± 0.2 degrees 2Θ. In some embodiments, the Oxil33 polymorph comprises, consists essentially of, or consists of Form B which produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 5.9, 11.9 and 17.96 0.2 degrees 2Θ in place of or in addition to the powder X-ray diffraction pattern of those above for Form B.

[0092] The ability to prepare salts depends on the acidity or basicity of a compound. Suitable salts of the compound include, but are not limited to, acid addition salts, such as those made with hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, maleic,

Petition 870180049765, of 11/06/2018, p. 48/129 / 108 fumaric, malic, tartaric, citric, benzoic, carbonic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, p-aminosenzoic-2-aminosulfonic and 2-aminosulfonic ; salts made with saccharin; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; and salts formed with organic or inorganic binders, such as quaternary ammonium salts. Additional suitable salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, cansilate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, stolate, stylate , fumarate, gluceptate, gluconate, glutamate, glycolylanarsanilate, hexyl resorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxinaftoate, iodide, isothioate, lactate, lactobionate, laurate, malate, maleate, mandelate, methylate, methylate, bromide, methylate, bromide , napsylate, nitrate, ammonium salt of N-methyl glucamine, oleate, pamoate (embonate), palmitate, pantothenate, phosphate diphosphate salts, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, tosylate trietiodide and valerate of the compounds.

[0093] In several modalities, Oxil33 includes one or more biological functions. That is, Oxil33 can induce a biological response when contacted with a mesenchymal stem cell or a bone marrow stromal cell. For example, Oxil33 can stimulate osteoblastic differentiation. In some embodiments, a bioactive composition including Oxil33 can include one or more biological functions when administered to a mammalian cell, for example, an in vitro cell or a cell in a human or an animal. For example, such a bioactive composition can stimulate osteoblastic differentiation. In some modalities, this biological function may arise from the stimulation of the hedgehog pathway.

Purification of Oxil33

Petition 870180049765, of 11/06/2018, p. 49/129 / 108 [0094] In some modalities, oxy-cholesterol, for example,

Oxil33, is highly purified. In some embodiments, Oxil33 can be crystallized or recrystallized. In some embodiments, the purified Oxil33 is formed by recrystallizing Oxil33 in a 3: 1 mixture of acetone / water, as shown below:

Layout 1

mw 420.67 g / m

θ27 ^Η 4β ° 3 ^H 2 ° mw 438.69 g / m [0095] As shown above, after crystallization, the purified Oxil33 forms a hydrate. However, in some modalities, Oxil33 is in anhydrous form. In some embodiments, the crystallinity percentage of any of the crystalline forms of Oxil33 described here may vary from the total amount of Oxil33.

[0096] In certain embodiments, Oxil33 can have a percentage crystallinity of a salt, hydrate, solvate or crystalline form of Oxil33 to be about at least 10%, at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. In some embodiments, the percent crystallinity can be substantially 100%, where substantially 100% indicates that the total amount of Oxil33 appears to be crystalline in the best way that can be determined using methods known in the art. Consequently, the therapeutically effective amounts of Oxil33 can include amounts that vary in crystallinity. These include examples in which an amount of Oxil33 crystallized in a solid form is subsequently dissolved, partially dissolved, or suspended or dispersed in a liquid.

Petition 870180049765, of 11/06/2018, p. 50/129 / 108 [0097] In one embodiment, the purified Oxil33 is crystallized as a monohydrate. However, in other embodiments, the purified Oxil33 can be crystallized in other hydrated forms, such as, for example, a dihydrate, hemihydrate, sesquihydrate, trihydrate, tetrahydrate or combinations thereof, as well as the corresponding solvated forms. In some modalities, Oxil33 is crystallized in an amorphous form. In other embodiments, the purified Oxil33 is crystallized as a co-crystal or a pharmaceutically acceptable salt.

[0098] In some modalities, oxy-cholesterol (for example, Oxil33) that can be used and can be in amorphous form and has a faster dissolution and release of the ATM, such as, for example, a release by rupture of the ATM of about from 10%, 15%, 20%, 25%, 30%, 35%, 45%, to about 50% of oxy-cholesterol for 24 or 48 hours.

[0099] In some embodiments, the non-purified Oxil33 can be solidified by mixing with heptanes. The product can subsequently be filtered and suspended in methylene chloride. In some embodiments, the unpurified Oxil33 can be filtered out of the suspension and crystallized using acetone and water or other organic or inorganic solvents (eg, diethyl ether, dichloromethane, ethyl acetate, acetone, n, n-dimethylformamide, acetonitrile , dimethylsulfoxide, ammonia, t-butanol, n-propanol, ethanol, methanol, acetic acid or a combination thereof).

[00100] In several modalities, the non-purified Oxil33 can be isolated and purified by any other traditional means. That is, the unpurified Oxil33 can be isolated and purified to the desired purity, for example, from about 95% to about 99.9% by filtration, centrifugation, distillation to separate volatile liquids based on their relative volatilities, crystallization , recrystallization, evaporation to remove volatile liquids from non-volatile solutes, solvent extraction to remove

Petition 870180049765, of 11/06/2018, p. 51/129 / 108 impurities, dissolving the composition in a solvent in which other components are soluble or other methods of purification.

[00101] In some embodiments, the purified Oxil33 is formed in crystalline form by crystallization, which separates the Oxil33 from the liquid feed stream by cooling the liquid feed stream or adding precipitants that reduce the solubility of by-products and unused reagents in the reaction mixture as soon as Oxil33 forms crystals. In some embodiments, the solid crystals are then separated from the remaining liquid by filtration or centrifugation. The crystals can be resolubilized in a solvent and then recrystallized and the crystals are then separated from the remaining liquid by filtration or centrifugation to obtain a highly pure sample of Oxil33. In some embodiments, the crystals can then be granulated to the desired particle size. In some embodiments, mineral particles (e.g., ceramic) can be used in ATM and can have a particle size of about 0.5 mm to about 1.5 mm. In some embodiments, the mineral particles can be from about 0.2 mm to about 0.5 mm.

[00102] In some embodiments, the unpurified Oxil33 can be purified when the purified Oxil33 is formed in the crystallized form in a solvent and then removed from the solvent to form a high purity of Oxil33 having a purity of about from 98% to about 99.99%. In some embodiments, Oxil33 can be recovered by filtration or vacuum filtration before or after purification.

Implants [00103] In some embodiments, the implant comprises an ATM that provides an acellular tissue structure for the cells to guide the tissue formation process in vivo in three dimensions in addition to oxy-cholesterol (for example, Oxil33). In some embodiments, the implant provides a porous structure to promote internal bone growth. The morphology

Petition 870180049765, of 11/06/2018, p. 52/129 / 108 of the ATM guides the migration of cells and the cells are able to migrate into or over the ATM. The cells are then able to proliferate and synthesize new tissues and form bones and / or cartilage. In some embodiments, one or more arrays of acellular tissue are stacked on top of each other. [00104] In some embodiments, the implant comprises an oxy-cholesterol uniformly arranged in a cohesive mass of an ATM. In certain embodiments, ATM is obtained from a human, porcine, goat, bovine, ovine or equine source. In other respects, TMJ is porcine collagen, while in other respects, TMJ is cross-linked porcine collagen. In many ways, the implant is a gel, paste or moldable paste and can be placed in a syringe. In certain aspects, the implant can be lyophilized and hydrated and then placed in a bone defect site by a surgeon or other responsible professional. In other respects, the lyophilized implant is hydrated and placed in a syringe for application to a bone defect site.

[00105] In some embodiments, the implant comprises bone material in addition to ATM and oxy-cholesterol. ATM can be obtained from a human or animal source and, in some cases, it can be non-cross-linked, in other cases, ATM can be cross-linked. In several cases, TMJ comprises acellular porcine collagen, which in some cases can be cross-linked. Each of the collagen, bone material and oxycholesterol of acellular porcine comprises particles that are mixed homogeneously with each other. In several modalities, the bone material comprises ATM of demineralized bone (DBM), ATM of mineralized bone, demineralized bone fibers, demineralized bone chips; and inorganic ceramics comprising tricalcium phosphate, hydroxyapatite, biphasic calcium phosphate, porous calcium phosphate ceramic or tricalcium phosphate, hydroxyapatite or a combination thereof. In some modalities, the implant also

Petition 870180049765, of 11/06/2018, p. 53/129 / 108 comprises an expandable phase or binder or swelling agent, for example, carboxyl cellulose or other cellulose derivatives.

[00106] The moldable implant, in some modalities, comprises a porous body that includes a particulate mineral material with an average particle diameter of about 0.4 mm to about 5.0 mm homogeneously mixed with an ATM. In some embodiments, mineral particles have an average particle size of about 0.5 mm to about 1.5 mm. In some embodiments, mineral particles have an average particle size of about 125 micrometers to about 750 micrometers.

[00107]

In some embodiments, particulate minerals (for example, TCP: HA) can be homogeneously arranged across the ATM with a particle size of about 0.10, 0.11, 0.12, 0.13, 0.14 , 0.15, 0.16, 0.17, 0.18, 0.19, 0.22, 0.22, 0.22, 0.23, 0.24, 0.25, 0.26, 0 , 27, 0.28, 0.29,

0.30, 0.31, 0.32, 0.38, 0.39, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0, 37, 0.38,

0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5 0, 0 , 51,

0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0, 64, 0.65,

0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0, 78, 0.79,

0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0, 92, 0.93,

0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, 1.25, 1.4, 1.5, 1.6, 1.7, 1, 8, 1.9, 2.0,

2.25 to about 2.5 mm. These particles can be in the form of granules, shavings, fibers or a combination thereof.

[00108] In several modalities, the particle size distribution of the ATM can be about 10 micrometers, 13 micrometers, 85 micrometers, 100 micrometers, 151 micrometers, 200 micrometers and all the sub-bands between them. In some embodiments, at least 75% of the particles are about 10 micrometers to 200 micrometers in size. In some embodiments, at least 85% of the particles are about 10 micrometers to 200 micrometers in size. In

Petition 870180049765, of 11/06/2018, p. 54/129 / 108 some modalities, at least 95% of the particles have a size of about 10 micrometers to about 200 micrometers. In some embodiments, all particles have a size of about 10 micrometers to about 200 micrometers. In some embodiments, at least 75% of the particles are about 20 micrometers to 180 micrometers in size. In some embodiments, at least 85% of the particles have a size of about 20 micrometers to about 180 micrometers. In some embodiments, at least 95% of the particles have a size of about 20 micrometers to about 180 micrometers. In some embodiments, all particles have a size of about 20 micrometers to about 180 micrometers.

[00109] In some embodiments, the one or more oxyesterols may, for example, have an average particle size of about 2.2 to about 10 microns. In some embodiments, the oxy-cholesterol particles have a minimum average particle size of about 2.2 microns, or about 2.5 microns, or about 3 microns, or about 4 microns. The particles can also have an average maximum particle size of about 10 microns, or about 8 microns, or about 7 microns, or about 5 microns. In some embodiments, oxy-cholesterol has a particle size of about 5 to 30 micrometers, or about 2 micrometers to about 20 micrometers, or 30 micrometers to 100 micrometers, however, in various modalities, variations of about 1 micrometer to 250 micrometers can be used. In some embodiments, oxy-cholesterol has a particle size of about 0.1 nm to about 1 micron to provide improved dissolution and faster implant release. In some embodiments, oxy-cholesterol (for example, Oxil33) is in the form of nanoparticles and about 10.0, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 , 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340,

Petition 870180049765, of 11/06/2018, p. 55/129 / 108

350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, at about 500 nm in diameter.

[00110] In some embodiments, oxy-cholesterol includes a particle size of about 0.1 mm to about 5 mm to lengthen the implant release time by reducing the dissolution rate of Oxil33 which can modulate bone formation. In addition, oxy-cholesterol particles can have a single-phase distribution. In addition, in some modalities, it may be useful to have a water-soluble oxy-cholesterol in order to produce an acute anti-inflammatory / analgesic effect that the implant is not providing.

[00111] In several embodiments, oxy-cholesterol is in the form of a polymorph, solvate, hydrate or a pharmaceutically acceptable salt. Oxy-cholesterol can alternatively be crystallized in an amorphous form. In some embodiments, oxy-cholesterol is in the form of a monohydrate. In some embodiments, oxy-cholesterol (eg, Oxil33) may be in amorphous form. In several embodiments, the implant comprises Oxil33 and an ATM in amorphous, crystalline or semi-crystalline form; where the crystalline form can include polymorphs, solvates or hydrates. Oxil33 includes polymorph of Form A, polymorph of Form B, polymorph of Form C, polymorph of Form D, polymorph of Form E, polymorph of Form F, polymorph of Form G, polymorph of Form H, polymorph of Form I or a mixture as described in US 15 / 082,695 and 15 / 374,610 incorporated herein by reference as if they were presented in full.

[00112] In some embodiments, an ATM of the present patent application includes an oxy-cholesterol in an amount from about 0.01 mg / cc to about 500 mg / cc. ATM can include oxycholesterol in an amount of about 10 mg / cc, 20 mg / cc, 25 mg / cc, 30 mg / cc, 40 mg / cc, 50 mg / cc, 60 mg / cc, 70 mg / cc cc, 80 mg / cc, 90 mg / cc, 100 mg / cc, 110 mg / cc, 120 mg / cc, 130 mg / cc, 140 mg / cc, 150 mg / cc, 160 mg / cc, 170 mg / cc, 180 mg / cc, 190 mg / cc, 200 mg / cc, 210 mg / cc, 220 mg / cc, 230 mg / cc, 240 mg / cc, 250 mg / cc, 260

Petition 870180049765, of 11/06/2018, p. 56/129 / 108 mg / cc, 270 mg / cc, 280 mg / cc, 290 mg / cc, 300 mg / cc, 310 mg / cc, 320 mg / cc, 330 mg / cc, 340 mg / cc, 350 mg / cc, 360 mg / cc, 370 mg / cc, 380 mg / cc, 390 mg / cc, 400 mg / cc, 410 mg / cc, 420 mg / cc, 430 mg / cc, 440 mg / cc, 450 mg / cc, 460 mg / cc, 470 mg / cc, 480 mg / cc, 490 mg / cc to about 500 mg / cc or any amount in between. In some embodiments, ATM releases from about 40 ng to about 5 mg of oxyesterol every hour.

[00113] In some embodiments, oxy-cholesterol can be loaded into the ATM in highly concentrated amounts. For example, in some embodiments, oxy-cholesterol is loaded into the ATM in an amount of at least 500 mg / cc. In some embodiments, oxyesterol is added to ATM in an amount from about 1 mg / cc to about 1 g / cc, from about 100 mg / cc to about 1 g / cc, from about 500 mg / cc at about 900 mg / cc, or from about 600 mg / cc to about 800 mg / cc. In other embodiments, oxy-cholesterol is added to ATM in an amount of about 500 mg / cc to about 600 mg / cc, about 600 mg / cc to about 700 mg / cc, about 700 mg / cc to about 800 mg / cc, about 800 mg / cc to about 900 mg / cc, or about 900 mg / cc to about 1 g / cc. In some embodiments, oxy-cholesterol is loaded in the ATM in an amount of about 134 mg / cc.

[00114] In some embodiments, oxy-cholesterol comprises a range of about 0.01% by weight to about 45% by weight based on the total weight of the TMJ or the implant before or after being moistened. In some embodiments, the implant comprises at least one biodegradable material at a weight% of about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32% , 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% or approximately 44% based on the total weight of the TMJ or implant.

[00115] In some embodiments, the ATM containing oxyesterol can have a release surface by rupture that releases around 10%, 15%,

Petition 870180049765, of 11/06/2018, p. 57/129 / 108

20%, 25%, 30%, 35%, 45%, at about 50% of oxycholesterol for more than 24 or hours.

[00116] In some embodiments, a high concentration of oxy-cholesterol can be loaded in the ATM and comprises from about 0.01% by weight to about 90% by weight of the ATM. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 0.1% by weight to about 90% by weight or from about 1.0% by weight to about 90% by weight of ATM. In some embodiments, the oxy-cholesterol can be loaded into the ATM in an amount of about 20% by weight to about 30% by weight, about 30% by weight to about 40% by weight, about 40% by weight to about 50% by weight, about 50% by weight to about 60% by weight, about 60% by weight to about 70% by weight, about 70% by weight to about 80% by weight, about from 80% by weight to about 90% by weight or about 90% by weight to about 99% by weight of ATM. In some embodiments, oxy-cholesterol can be loaded into the ATM in an amount of about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or about 95 % by weight of ATM.

[00117] In some modalities, ATM releases oxycholesterol over a period of 1-90 days, 1-10 days, 1-3 days, 3-7 days, 3-12 days, 3-14 days, Ί10 days 7- 14 days, 7-21 days, 7-30 days, 7-50 days, 7-90 days, 7-140 days, 14140 days, 3 days to 135 days, 3 days to 180 days, or 3 days to 6 months. In some embodiments, bone growth will be observed over a period of at least 14 days, for example, 14-90 days, 14-30 days, 14-60 days, 21-90 days, 21-180 days; 14-210 days, or 14 days to 6 months.

[00118] In some embodiments, ATM (in some cases, acellular porcine collagen) comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 ,

Petition 870180049765, of 11/06/2018, p. 12 589/ 108

30.0, 30.5, 31.0, 31.5, 32.0, 32.5, 33.0, 33.5, 34.0, 34.5, 35.0, 35.5, 36, 0, 36.5, 37.0, 37.5, 38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44.0, 44.5, 45.0, 45.5, 46.0, 46.5, 47.0, 47.5, 48.0, 48, 5, 49.0, 49.5 or about 50.0% w / w, w / v or v / v of the implant. In certain embodiments, ATM is acellular porcine collagen, which can be present in an amount of about 0.1% by weight to about 25% by weight of the implant. In some embodiments, ATM is present in an amount between about 0.01% by weight to about 50% by weight, about 0.1% by weight to about 20% by weight or about 0.1% by weight to about 25% by weight of the implant. In some embodiments, ATM is cross-linked acellular porcine collagen, which can be present in an amount of about 0.1% by weight to about 25% by weight, about 1.0% by weight at about 20 % by weight, about 20% by weight to about 30% by weight, about 30% by weight to about 40% by weight or about 40% by weight to about 50% by weight.

[00119] In some embodiments, the implant further comprises a fluid of about 25% by weight to about 50% by weight that is added to improve the malleability of the moldable implant. The malleable implant is configured to be moldable to any shape desired to fit into a bone defect site. In some embodiments, the malleable implant can be shaped to fit a surgical site, such as a bone defect site. The shape of the ATM can be adapted to the place where it is to be located. For example, it can take the form of a piece, a plug, a pin, a pin, a cylinder, a block, a wedge, a sheet, a strip, etc. The term "shape" refers to a determined or regular shape or configuration in contrast to an indeterminate or vague shape or configuration (as in the case of a lump or other solid mass with no special shape) and is characteristic of such materials as sheets, strips , plates, discs, cores, tubes, wedges, cylinders or the like. This includes shapes ranging from regular geometric shapes to irregular shapes, angular or not

Petition 870180049765, of 11/06/2018, p. 59/129 / 108 geometric or feature combinations with any of these characteristics. In some modalities, the implant is malleable before being implanted in a surgical site. In such modalities, a medical professional can shape the implant into the desired shape and allow the implant to heal or dry before the implant. In such modalities, a doctor can shape the implant directly into a bone defect site. The implant is malleable and configured to be pressed into a bone defect site to fill the cracks in a bone defect site. In some embodiments, the implant is malleable when moistened and is configured to remain malleable while in contact with a bone defect site. In certain embodiments, the moldable implant is placed in a syringe for application to a bone defect site. The syringe containing the implant can also be placed in a vacuum sealed bag.

[00120] In some modalities, the malleable ATM can be formed to fit in the empty space of an intercorporeal cage or around the outside of the cage in the intervertebral space.

[00121] The implant can be moistened or hydrated with a variety of fluids to form a malleable and moldable implant or an aqueous paste that can be subsequently lyophilized. In some modalities, the TMJ is moistened with sterile water, physiological saline, sodium chloride, dextrose, lactated Ringer's solution, phosphate-buffered saline (PBS), blood, bone marrow aspirate, bone marrow fractions or a combination thereof. The amount of fluid with which the ATM can be moistened includes about 0.25, 0.5, 0.75,

Petition 870180049765, of 11/06/2018, p. 60/129 / 108

38.0, 38.5, 39.0, 39.5, 40.0, 40.5, 41.0, 41.5, 42.0, 42.5, 43.0, 43.5, 44, 0, 44.5, 45.0, 45.5.46.0, 46.5.47.0, 47.5, 48.0.48.5, 49.0.49.5 to about 50, 0 ml.

[00122] In some modalities, the implant is hydrated with hyaluronic acid, cellulose ethers (such as carboxytylcellulose), collagen, gelatin, autoclaved bone powder, osteoconductive transporters, whole blood, blood fractions, bone marrow aspirate, aspirated from concentrated bone marrow and mixtures thereof. Non-limiting examples of blood fractions include serum, plasma, platelet rich plasma (PRP), concentrated platelet rich plasma, platelet poor plasma and concentrated platelet poor plasma. After hydration, the implant becomes a gel, a paste or a paste that can be shaped into a predetermined shape in about 30 seconds or administered to a bone defect and manipulated to conform to the bone defect in such a way as to promote healing .

[00123] In some embodiments, the implant comprises a porous ATM configured to allow the influx of at least bone and / or cartilage cells. In some embodiments, ATM is also configured to release an active agent, such as oxy-cholesterol. By "porous", it is understood that the ATM has a plurality of pores. The pores of the TMJ are large enough to allow the influx of blood, other body fluid, and progenitor and / or bone cells and / or cartilage inside to guide the tissue formation process in vivo in three dimensions .

[00124] In some embodiments, the ATM comprises a plurality of pores. In some modalities, at least 10% of the pores are between about 50 micrometers and about 500 micrometers at their widest points. In some modalities, at least 20% of the pores are between about 50 micrometers and about 250 micrometers at their widest points. In some modalities, at least 30% of the pores are between about 50 micrometers and about 150 micrometers in their

Petition 870180049765, of 11/06/2018, p. 61/129 / 108 widest points. In some modalities, at least 50% of the pores are between about 10 micrometers and about 500 micrometers at their widest points. In some modalities, at least 90% of the pores are between about 50 micrometers and about 250 micrometers at their widest points. In some modalities, at least 95% of the pores are between about 50 micrometers and about 150 micrometers at their widest points. In some modalities, 100% of the pores are between about 10 micrometers and about 500 micrometers at their widest points.

[00125] In some embodiments, the ATM has a porosity of at least about 30%, at least about 50%, at least about 60%, at least about 70%, at least about 90%, at least about 95%, or at least about 99%. The pores can support cell growth, formation or remodeling of bone, cartilage and / or vascular tissue. [00126] In some modalities, oxy-cholesterol is administered in an implant that is in solid or semi-solid form. In several respects, oxy-cholesterol is (3S, 5S, 6S, 8R, 9S, 10R, 13S, 14S, 17S) 17 - ((S) -2-hydroxyoctan-

2-yl) -10, 13-dimethyl-hexadecahydro-1 H-cyclopenta [a] phenanthrene-3,6-diol (Oxil33), Oxil49, Oxil53, Oxil54, Oxil55 or hydrates, solvates, amorphous forms, polymorphs or their salts pharmaceutically acceptable. In other respects, oxycholesterol includes polymorph of Form A, polymorph of Form B, polymorph of Form C, polymorph of Form D, polymorph of Form E, polymorph of Form F, polymorph of Form G, polymorph of Form H, polymorph of Form I of Oxil33 or a mixture thereof as described in US documents no. serial number 15 / 082,695 and 15 / 374,610 incorporated herein by reference as if they were presented in full.

[00127] The moldable shape of the implant can have a pre-set viscosity in the range of about 1 to about 2000 centipoise (cps), 1 to about 200 cps, or 1 to about 100 cps. In several modalities, the implant

Petition 870180049765, of 11/06/2018, p. 62/129 / 108 semisolid or solid can comprise an acellular porcine collagen with a molecular weight (MW), as shown by the inherent viscosity (IV) of about 0.10 dL / g to about 1.2 dL / g or about 0.20 dL / g about 0.50 dL / g. Other IR ranges include, but are not limited to, about 0.05 to about 0.15 dL / g, about 0.10 to about 0.20 dL / g, about 0.15 to about 0 , 25 dL / g, about 0.20 to about 0.30 dL / g, about 0.25 to about 0.35 dL / g, about 0.30 to about 0.35 dL / g , about 0.35 to about 0.45 dL / g, about 0.40 to about 0.45 dL / g, about 0.45 to about 0.55 dL / g, about 0, 50 to about 0.70 dL / g 0.55 to about 0.6 dL / g, about 0.60 to about 0.80 dL / g, about 0.70 to about 0.90 dL / g, about 0.80 to about 1.00 dL / g, about 0.90 to about 1.10 dL / g, about 1.0 to about 1.2 dL / g, about 1.1 to about 1.3 dL / g, about 1.2 to about 1.4 dL / g, about 1.3 to about 1.5 dL / g, about 1.4 to about 1.6 dL / g, about 1.5 to about 1.7 dL / g, about 1.6 to about 1.8 dL / g, about 1.7 to about 1.9 dL / g, or about 1.8 to about 2.1 dL / g.

[00128] In some modalities, the ATM has an elasticity modulus in the range of about ¹ x ^{10 2} to about 6x10 ⁵ dynes / cm ² , or ² x 10 ⁴ to about 5x10 ⁵ dynes / cm ² , or 5x10 ⁴ to about 5x10 ⁵ dynes / cm ² . In some modalities, ATM is in lyophilized form.

[00129] In some embodiments, ATM has a density between about 1.6 g / cm ³ , and about 0.05 g / cm ³ . In some embodiments, ATM has a density of between about 1.1 g / cm ³ , and about 0.07 g / cm ³ . For example, the density can be less than 1 g / cm ³ , less than about 0.7 g / cm ³ , less than about 0.6 g / cm ³ , less than about 0.5 g / cm ³ , less than about 0.4 g / cm ³ , less than about 0.3 g / cm ³ , less than about 0.2 g / cm ³ , or less than about 0.1 g / cm ³ .

[00130] In some embodiments, the diameter or diagonal of the ATM can vary from 1 mm to 50 mm. In some embodiments, the diameter or diagonal of the ATM can vary from 1 mm to 30 mm, or 5 mm to 10 mm, which is

Petition 870180049765, of 11/06/2018, p. 63/129 / 108 small enough to fit through an endoscopic cannula, but large enough to minimize the number of matrices needed to fill a large bone defect (for example, osteochondral defect). In some modalities, at the time of surgery, the TMJ may be shaped by the surgeon in the desired shape to fit the tissue or bone defect.

[00131] In some embodiments, the porous interior may contain oxy-cholesterol within the ATM and because the interior is porous, the oxy-cholesterol is uniformly distributed throughout the ATM when the oxy-cholesterol is incorporated in the ATM, as discussed here.

[00132] In some modalities, oxy-cholesterol will be kept inside the TMJ and released into the environment around the TMJ (for example, bone defect, osteochondral defect, etc.) as the TMJ degrades over time.

[00133] In some modalities, TMJ can be seeded with harvested bone cells and / or bone tissue, such as, for example, cortical bone, autogenous bone, halogenic bones and / or xenogenic bone. In some modalities, TMJ can be seeded with harvested cartilage cells and / or cartilage tissue (for example, autogenous, halogenic and / or xenogenic cartilage tissue). For example, before insertion into the target tissue site, the TMJ can be moistened with the graft tissue / bone cells, usually with cells / bone tissue aspirated from the patient, in a ratio of about 3: 1, 2: 1, 1: 1, 1: 3 or 1: 2 by volume. The cells / bone tissue can penetrate the supplied TMJ, and the TMJ can be kneaded manually or with a machine, thus obtaining a flexible and cohesive consistency that can subsequently be packed into the bone defect. In some embodiments, ATM provides a malleable, non-water-soluble conveyor that allows precise placement and retention at the implantation site. In some embodiments, bone and / or cartilage cells

Petition 870180049765, of 11/06/2018, p. 64/129 / 108 collected can be mixed with oxy-cholesterol and sown inside the ATM.

Acelular Tissue Matrix (ATM) [00134] As used here, an “acellular tissue matrix” (ATM) is a tissue-derived structure that is made from any of a wide range of tissues containing collagen removing all, or substantially all viable cells and also all detectable dead cells, subcellular components and / or debris generated by dead or dying cells. In many respects, an “acellular tissue matrix” is a matrix that: (a) is made of any of a wide variety of collagen-based tissue; (b) is acellular; and (c) it retains the biological and structural functions possessed by the native tissue or organ from which it was made. As used here, an ATM without "substantially all viable cells" is an ATM where the concentration of viable cells is less than 1% (for example, less than: 0.1%; 0.01%; 0.001%; 0 , 0001%; 0.00001%; or 0.000,001%) than in the tissue or organ from which the bone material was made. An ATM “substantially without dead cells and / or cellular debris” is one that contains less than 10% (for example, less than: 8%; 5%; 1%; 0.1%; 0.001%; 0.0001%; or less) of dead cells and / or cell debris present in bone material after a cell removal process.

[00135] The biological functions retained by ATM include cell recognition and cell binding, as well as the ability to support cell dissemination, cell proliferation and cell differentiation. These functions are provided by non-denatured collagen proteins (eg Type I collagen) and a variety of non-collagen molecules (eg proteins that serve as binders for molecules such as integrins, molecules with a high charge density, such as glycosaminoglycans ). for example, hyaluronan) or proteoglycans, or other adhesions). The structural functions retained by useful acellular matrices

Petition 870180049765, of 11/06/2018, p. 65/129 / 108 include the maintenance of histological architecture, the maintenance of the three-dimensional matrix of tissue components and physical characteristics such as strength, elasticity and durability, defined porosity and macromolecule retention. The efficiency of the biological functions of an ATM can be measured, for example, by its ability to support cell proliferation and is at least 50% (for example, at least: 50%; 60%; 70%; 80%; 90 %; 95%; 98%; 99%; 99.5%; 100%, or more than 100%) of the native tissue or organ from which the ATM is manufactured. In addition, the integrity of the basement membrane at the ATM, measured by electron microscopy and / or immunohistochemistry, is at least 70% of the membrane of the native tissue or organ from which the ATM is made.

[00136] Although the properties described above are retained by ATM, bone material can be produced from any mammalian collagen-based tissue (eg, dermis, fascia, umbilical cords, placentas, heart valves, ligaments, tendons, vascular tissue (arteries and veins, such as saphenous veins, neural connective tissue or ureters). In some respects, the tissues in which these allografts can be placed include essentially any tissue that can be remodeled by invasion or infiltration of cells. Relevant tissues include skeletal tissues such as cartilage, ligaments, fascia and tendon Other tissues in which any matrix of the above acellular tissue can be placed include, without limitation, skin, gum, dura mater, myocardium, vascular tissue, neural tissue, striated muscle , smooth muscle, bladder wall, ureter tissue, intestine and urethral tissue.

[00137] In various embodiments, the acellular tissue matrix material can comprise any tissue containing collagen and material of mammalian, human or animal origin. The matrix material of the acellular tissue may be a tissue predominantly comprising type I collagen. In other respects, the starting materials include dermis and tendons. In some embodiments, porcine tissue materials are processed to provide

Petition 870180049765, of 11/06/2018, p. 66/129 / 108 collagenous material, although it is understood that other mammalian sources may alternatively be employed, such as, for example, primates, cows, sheep, goats or horses. An example of an acellular porcine tissue matrix useful for implanting this application is Permacol®. Permacol® is derived from the porcine dermis that has undergone the removal of cellular components and genetic material. The remaining extracellular acellular tissue (ATM) matrix, mainly collagen, is then cross-linked to form an ATM that contains 20% collagen in 80% saline. Permacol® has been found to induce minimal foreign body rejection and is resistant to biodegradation by native collagenases.

[00138] Depending on the starting material, collagen particles may contain a proportion of elastin. Thus, the particles and coherent collagenous materials formed therefrom essentially consist of collagen optionally with small proportions of elastin.

[00139] Acellular collagen particles can be formed using any suitable process. The architecture of the original collagen fiber and the molecular ultrastructure of the natural tissue must be retained in the particles. Collagenous tissue is not solubilized or denatured in the process, so its natural fiber structure is maintained. All that is needed is that the steps used in its production result in matrices with the biological and structural properties described above. Useful production methods include those described in US Patents 5,397,353 and 6,936,271, published US patent applications 2013/0045923 and 2013/0267465, and EP1112096, all of which are incorporated herein by reference in their entirety.

[00140] Thus, freshly cut natural tissue material can be treated to remove substantially all of the lipids and lipid residues therefrom and are then treated to remove proteins from non-fibrous tissues and cellular elements. Lipid extraction can be achieved by solvent extraction using an organic solvent, such as

Petition 870180049765, of 11/06/2018, p. 67/129 / 108 as acetone. Other non-limiting examples of suitable solvents include non-aqueous solvents such as ethanol and ether.

[00141] Non-fibrous tissue proteins include glycoproteins, proteoglycans, globular proteins and the like, such as components of extracellular ATM. Cellular elements include antigenic proteins and enzymes and other cellular debris that arise from processing conditions. These portions of the natural tissue material can be removed by treatment with a proteolytic enzyme, such as trypsin. It has been previously verified that above 20 ° C treatment with trypsin can, in some cases, result in a change in the structure of the collagen fiber which leads to less physical resistance. In addition, low temperatures favor the growth of microorganisms in the preparation. Therefore, it is useful to carry out trypsin treatment at a temperature below 20 ° C. In addition, trypsin is more stable below 20 ° C and smaller amounts may be required. Any suitable trypsin concentration can be used, for example, a concentration within the range of about 0.01 g / 1 to 25 g / 1. It was found that good results can be obtained using about

2.5 g / 1 trypsin. Optionally, additional treatments can be carried out, such as treatment with one or more additional enzymes, for example, a carbohydrate dividing enzyme.

[00142] The resulting collagenous material is then reduced to particles, care being taken to ensure that size reduction is not associated with a degradation of the original collagen fiber architecture and molecular ultrastructure of the starting material. The particles can be produced by grinding or grinding using, for example, a ball or hammer mill, which can be cooled to an appropriate temperature. The sheet material can be cut into small pieces before grinding. The grinding can be carried out in dry form (moisture content less than 10%) or in frozen hydrated form (moisture content from 20 to 80%).

Petition 870180049765, of 11/06/2018, p. 68/129 / 108 [00143] The acellular collagen particles can be of any suitable size. In some embodiments, the acellular collagen particles have an average diameter within the range of about 5 pm to about 1000 pm and, in other embodiments, from about 50 pm to about 500 pm. Good results were obtained using acellular collagen particles with an average diameter of approximately 150 pm.

[00144] In summary, the steps involved in the production of an ATM include the collection of tissue from a donor (for example, a human corpse or any of the mammals listed above, including porcine dermis), chemical treatment in order to stabilize the tissue and prevent biochemical and structural degradation in conjunction with, or followed by, removal of cells under conditions that, similarly, retain biological and structural function. The bone material can optionally be treated with a cryopreserving and cryopreserving agent and, optionally, lyophilized, again under conditions necessary to maintain the described biological and structural properties of ATM. After freezing or lyophilization, the tissue can be fragmented, for example, pulverized or micronized to produce a particulate ATM under similar conditions of conservation of function. All steps are generally performed under sterile aseptic conditions.

[00145] An exemplary ATM production method, which is described in more detail in US Patent 5,397,353, contains the following steps: (a) the freshly cut dermis is extracted with acetone with one or more changes; (b) the dermis is placed in buffer or saline to remove acetone; (c) the dermis is then subjected to digestion with trypsins in a solution of pH 7.0 to 9, to remove antigenic proteins and cellular elements such as hair follicles and sweat glands; (d) optionally, the tissue can be treated with a carbohydrate-dividing enzyme, such as amylase, hyaluronidase or neuramidase to remove antigenic polysaccharides and mucopolysaccharides; (e) the purified tissue is stabilized by

Petition 870180049765, of 11/06/2018, p. 69/129 / 108 treatment with a diisocyanate, for example, hexane diisocyanate as a 0.1% solution in acetone; (f) two more washes with acetone; (g) rinse with buffer or saline; (h) storage in the presence of a bactericide or sterilization with gamma irradiation; and (i) packaging under sterile conditions. According to this procedure, a substantially non-antigenic and non-resorbable collagenous fibrous tissue preparation of human or animal origin is provided, which is suitable for homo or heterotransplantation as a permanent repair for bone damage, the preparation of which retains the natural structure and original architecture of human or animal tissue, is substantially free of proteins and glycoproteins of non-fibrous tissue, is substantially free of cellular elements, is substantially free of lipids and lipid residues and is not cytotoxic, in which the preparation, when implanted, is capable to be recolonized by host cells (eg, osteoblasts, osteoclasts, etc.) and revascularized. In addition, a substantially non-antigenic and non-resorbable collagenous fibrous tissue preparation is also substantially free of antigenic polysaccharides and mucopolysaccharides.

[00146] In accordance with an additional aspect of the present application, a process is provided for the manufacture of an acellular collagenous material from a natural tissue material, wherein the process comprises the steps of: (i) treating the material of natural fabric with an organic solvent; (ii) treating the natural tissue material with a proteolytic enzyme, so as to provide a treated material that is substantially free of non-fibrous tissue proteins, cellular elements and lipids or lipid residues and exhibits original collagen fiber architecture and molecular ultrastructure the tissue material from which it is derived; (iii) reducing the treated material to a plurality of collagen particles comprising fragments of collagen fibers; and (iv) lyophilize the collagen particles suspended in a

Petition 870180049765, of 11/06/2018, p. 70/129 / 108 lyophilization suspension, in which the process, in some aspects, excludes the swelling of collagen particles with acid and / or alkali.

[00147] According to another aspect of the present application, a process is provided for the manufacture of an acellular collagenous material from a plurality of collagen particles, the collagen particles comprise fragments of collagen fibers and being derived from a material treated, the treated material having been produced from a natural tissue material by treatment with an organic solvent and with a proteolytic enzyme in order to provide a material that is substantially free of non-fibrous tissue proteins, cellular elements and lipids or residues lipids and exhibit molecular ultrastructure and original collagen fiber architecture of natural tissue material, in which the process comprises the step of lyophilization of the collagen particles suspended in a lyophilized suspension medium and in which the process excludes the swelling of the collagen particles with acid and / or alkali.

[00148] In some embodiments, the tissue will infiltrate bone material to a degree of at least 50 percent within about 1 month to about 6 months after TMJ implantation. In other modalities, about 75 percent of bone material will be infiltrated by tissue within about 2-3 months after TMJ implantation. In some cases, the ATM will be substantially about 90 percent or more, submerged or surrounded by tissue within about 6 months after implantation of the ATM. In other cases, the TMJ will be completely submerged or surrounded by tissue within about 9-12 months after implantation.

[00149] In some respects, the implant of the present application may include an oxy-cholesterol uniformly disposed in an ATM, in which the ATM comprises acellular collagen from other sources, for example, primates, cattle, sheep or fish, in some cases, reticulated, while in others not cross-linked. This type of ATM can be placed in a syringe to

Petition 870180049765, of 11/06/2018, p. 71/129 / 108 application in a surgical site or can be lyophilized for rehydration in a surgical site. Useful acellular tissue arrays prepared from sources other than porcine collagen, as described above, can also be used to form an implant in which an oxy-cholesterol is uniformly disposed (e.g., dispersed). Implants prepared from these ATMs can also include other bone material including inorganic particles with or without binding or swelling materials. As with porcine collagen ATMs, in these ATMs, collagen can be cross-linked or non-cross-linked. Implants, including ATMs prepared from non-porcine acellular collagen, can be administered in the same way as ATMs containing acellular porcine collagen, namely through a syringe, or they can be lyophilized and then rehydrated for application in a site. surgical.

[00150] In some embodiments, the implant prepared from a substantially non-antigenic collagenous fibrous tissue preparation sheet described above can be derived from porcine dermis. In some embodiments, the acellular collagenous tissue sheet of tissue fibers derived from the porcine dermis can be cross-linked by means of a polyisocyanate, which in some cases is hexamethylene diisocyanate (HMDI). A useful product for the implant described in this application is Permacol®, a biological material prepared from the porcine dermis and available as a paste. The porcine dermis is a good source of acellular collagen, as it supports the infiltration and revascularization of the host cell. Since Permacol® requires minimal rehydration, an implant containing Permacol® and oxy-cholesterol can be prepared in just 30 seconds. Permacol® is typically cross-linked with diisocyanate, which makes it quite resistant to enzymatic degradation.

[00151] In other modalities, an implant is provided that comprises a biocompatible carrier having dispersed in the same

Petition 870180049765, of 11/06/2018, p. 72/129 / 108 solid or semi-solid particles of acellular collagenous material derived from a natural tissue material, including porcine dermis, in which the acellular collagenous material retains the original fiber architecture and molecular ultrastructure of the natural tissue material from which it is derived, where the acellular collagenous material is substantially free of non-fibrous tissue proteins, glycoproteins, cell elements, lipids or lipid residues, where the acellular collagenous material is not cytotoxic, where the implant composition is capable of being used as a component of a paste, gel or an injectable solution, and in which the particles of acellular collagenous material have a particle size within the range of approximately 50 microns to approximately 500 microns.

[00152] In other embodiments, the particles of acellular collagenous material have a particle size distribution in which the particle sizes of at least 50 percent of the particles are within 35 percent of the average particle size, can be free of antigenic polysaccharides and mucopolysaccharides, and may be substantially free of antigenic polysaccharides and mucopolysaccharides. In various embodiments, the acellular collagenous material contains a proportion of elastin. In other respects, the acellular collagenous material is cross-linked. In certain implementations, the biocompatible carrier is at least one of saline, glycerol, a dextran solution, a non-toxic antigenic viscous polysaccharide or a combination thereof. In certain aspects, the acellular collagenous material comprises approximately 10 weight percent to approximately 90 weight percent of the implant composition.

[00153] In some modalities, the ATM comprises acellular collagen-containing biomaterials that, when placed in a bone defect, provide structures around which the patient's new bone and / or cartilage will grow, gradually replacing the ATM transporter as the target site heals. Examples of suitable carrier matrices can

Petition 870180049765, of 11/06/2018, p. 73/129 / 108 include, but are not limited to, the ATM MasterGraft® produced by Medtronic Sofamor Danek, Inc., Memphis, Tenn .; MasterGraft® grease produced by Medtronic Sofamor Danek, Inc., Memphis, Tennessee; Absorbable Collagen Sponge (“ACS”) produced by Integra LifeSciences Corporation, Plainsboro, N.J .; collagen fibers of bovine skin coated with hydroxyapatite, for example, Healos® sold by Johnson & Johnson, USA; collagen sponges, for example, Hemostagene® marketed by Coletica SA, France, or for example, Helisat® marketed by Integra Life Sciences Inc., USA; and Collagraft® bone graft ATM produced by Zimmer Holdings, Inc., Warsaw, Ind.

[00154] In some embodiments, the acellular collagen contains both soluble acellular collagen and insoluble acellular collagen fibers. Soluble acellular collagen and insoluble acellular collagen fibers can first be prepared separately and then combined. Both soluble acellular collagen and insoluble acellular collagen fibers can be derived from a variety of sources, including human, bovine, sheep, fish, or porcine sources.

[00155] In certain embodiments, the acellular collagen ATM includes moldable compositions that include insoluble acellular collagen fibers at a level of 0.04 g / cc to 0.1 g / cc of the ATM, and soluble collagen at a level of 0.01 g / cc to 0.08 g / cc of the ATM. In other embodiments, such compositions include insoluble acellular collagen fibers at a level of about 0.05 to 0.08 g / cc of ATM and soluble acellular collagen at a level of about 0.02 to about 0.05 g / cc of ATM. In general, ATM will include insoluble acellular collagen fibers in an amount (weight percentage) that is at least greater than or equal to the amount of soluble acellular collagen to contribute beneficially to the desired handling and implant properties of the ATM material. In some embodiments, the acellular collagenous ATM will include insoluble acellular collagen and acellular collagen fibers

Petition 870180049765, of 11/06/2018, p. 74/129 / 108 soluble in a weight ratio of 4: 1 to 1: 1, in some cases, from about 75:25 to about 60:40. In other embodiments, the acellular collagen ATM may include insoluble acellular collagen fibers and soluble acellular collagen fibers in a weight ratio of about 75:25 to about 65:35, and in an embodiment about 70:30.

[00156] In some embodiments, the acellular collagen ATM comprises acellular porcine collagen and / or a non-polymeric material. In some embodiments, the acellular collagen ATM may include a biodegradable biopolymer that can provide immediate or prolonged release of oxy-cholesterol. For example, acellular porcine collagen ATM may comprise polyether ether ketone (PEEK). In some embodiments, the acellular collagen ATM may comprise one or more of poly (alpha-hydroxy acids), polyglycolide (PG), polyethylene glycol (PEG), poly (alpha-hydroxy acid) conjugates, poly-polyesters (POE), polyaspirins , polyphosphages, collagen, hydrolyzed collagen, gelatin, hydrolyzed gelatin, hydrolyzed gelatin fractions, elastin, starch, pregelatinized starch, hyaluronic acid, chitosan, alginate, albumin, fibrin, vitamin E analogues, such as alpha-tocopheryl acetate, succinate dalpha-tocopheryl, D, L-lactide or L-lactide, caprolactone, dextrans, vinylpyrrolidone, polyvinyl alcohol (PVA), PVA-g-PLGA, PEGT-PBT (polyactive) copolymer, methacrylates, PEO-PPO- copolymers PAA, PLGAPEO-PLGA, PEG-PLG, PLA-PLGA, poloxamer 407, triblock copolymers PEG-PLGA-PEG, POE, SAIB (sucrose isobutyrate acetate), polydioxanone, methylmethacrylate (MM A), MM A and N-vinylpyrrolidone , polyamide, oxycellulose, gl acid acid copolymer icolic and trimethylene carbonate, polyesteramides, polyether ether ketone, polymethylmethacrylate, silicone, hyaluronic acid, chitosan or combinations thereof.

[00157] In some modalities, the implant may not be completely biodegradable. For example, the device can understand

Petition 870180049765, of 11/06/2018, p. 75/129 / 108 polyurethane, polyurea, polyether (amide), PEBA, thermoplastic elastomeric olefin, copolyester and styrenic thermoplastic elastomer, steel, aluminum, stainless steel, titanium, metal alloys with a high content of non-ferrous metals and a low relative proportion of iron, carbon device, glass device, plastic, ceramics, methacrylates, poly (Nisopropylacrylamide), PEO-PPO-PEO (pluronic) or combinations thereof. In some embodiments, these types of matrices may need to be removed after a certain period of time.

[00158] In some embodiments, the implant comprises at least one acellular porcine collagen comprising one or more of poly (lactide-co-glycolide) (PLGA), polylactide (PLA), polyglycolide (PGA), D-lactide, D, L-lactide, L-lactide, D, L-lactide-co-8-caprolactone, Llactida-co-8-caprolactone, D, L-lactide-co-glycolide-co-8-caprolactone, poly (D, L- lactide-co-caprolactone), poly (L-lactide-co-caprolactone), poly (Dlactide-co-caprolactone), poly (D, L-lactide), poly (D-lactide), poly (L-lactide), poly (stereo) or a combination thereof. In some embodiments, oxy-cholesterol is encapsulated in an acellular porcine collagen.

[00159] In some embodiments, the ATM comprises one or more polymers (for example, PLA, PLGA) with a PM of about 15,000 to about 150,000 Da or from about 25,000 to about 100,000 Da.

[00160] In some embodiments, the implant comprises at least one biodegradable material in a weight% of about 99.5%, 99%, 98%,

97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%,

84%, 83%, 82%, 81%, 80%, 79%, 78%, 76%, 75%, 74%, 73%, 72%, 71%,

70%, 65%, 60%, 55%, 50%, 45%, 35%, 25%, 20%, 15%, 10%, or about

5% based on the total weight of the TMJ or implant. In some embodiments, the acellular porcine collagen comprises a range of about 0.1% to about 20% based on the total weight of the TMJ or the implant. In some embodiments, the acellular porcine collagen comprises a range

Petition 870180049765, of 11/06/2018, p. 76/129 / 108 of about 0.1% to about 15% based on the total weight of the ATM or implant. In some embodiments, the acellular porcine collagen comprises about 14%, 13%, 12%, 11%, 9%, 8%, 7%, 6% or about 5% based on the total weight of the ATM or the implant.

[00161] In some embodiments, acellular porcine collagen is present in the ATM in an amount of about 0.01% by weight to about 50% by weight or from about 8.0% by weight to about 50% by weight of the ATM. In some embodiments, the acellular porcine collagen is present in an amount of about 0.1% by weight to about 10% by weight, from about 10% by weight to about 20% by weight, from about 20 % by weight to about 30% by weight, from about 30% by weight to about 40% by weight, or from about 40% by weight to about 50% by weight.

[00162] Mannitol, trehalose, dextran, mPEG and / or PEG can be used as a plasticizer for the polymer. In some embodiments, the polymer and / or plasticizer can also be coated on the implant to provide the desired release profile. In some embodiments, the thickness of the coating may be thin, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 microns for thicker coatings of 60, 65, 70, 75 , 80, 85, 90, 95, 100 microns to delay the release of oxy-cholesterol from the implant. In some embodiments, the range of coating on the implant ranges from about 5 microns to about 250 microns or from 5 microns to about 200 microns to delay implant release.

[00163] Compressive strength may be desirable for many tissue engineering applications, such as tibial plateau fractures, acetabular deficiency, comminuted long bone fractures, oral maxillofacial defects, spine fusions, and subchondral cartilage defects. Compression-resistant collagen matrices will help to favor adequate volumes of newly formed bone.

Petition 870180049765, of 11/06/2018, p. 77/129 / 108 [00164] In some embodiments, the ATM is resistant to compression, where the ATM resists reduction in size or an increase in density when a force is applied in comparison with collagen matrices without the bone material disposed therein. . In various modalities, the acellular collagen TMJ resists compression by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 %, 75%, 80%, 85%, 90%, 95% or more in one or all directions when a force is applied to the ATM.

Bone Material [00165] In some modalities, the implant may contain demineralized bone material arranged in it. The demineralized bone material may comprise demineralized bone powder, shavings, triangular prisms, spheres, cubes, cylinders, fragments, fibers or other shapes with irregular or random geometries. These may include, for example, "substantially demineralized", "partially demineralized" or "completely demineralized" cortical and spongy bone. These also include surface demineralization, where the surface of the bone construct is substantially demineralized, partially demineralized or completely demineralized, but the body of the bone construct is completely mineralized. In some embodiments, the acellular tissue matrix may comprise some completely mineralized bone material. The configuration of the bone material can be achieved by grinding, cutting, cutting or machining the entire bone as described, for example, in US Patent 5,899,939. All disclosure is incorporated herein by reference in this disclosure.

[00166] In some embodiments, the bone material may comprise elongated demineralized bone fibers with an average length to average thickness ratio or fiber aspect ratio of about 50: 1 to about 1,000: 1. In general appearance, elongated demineralized bone fibers can be round, spherical, granular, elongated, powders, shavings,

Petition 870180049765, of 11/06/2018, p. 78/129 / 108 fibers, cylinders, threads, narrow strips, thin sheets or a combination thereof. The elongated demineralized bone fibers can have a substantially linear appearance or can be rolled up to resemble springs. In some embodiments, elongated demineralized bone fibers are irregularly shaped, including, for example, linear, serpentine or curved shapes. The elongated bone fibers can be demineralized, however, part of the original mineral content can be retained when desirable for a particular modality. In some embodiments, the bone material comprises elongated demineralized bone fibers and shavings. In some embodiments, the bone material comprises completely demineralized fibers and demineralized surface shavings. In some embodiments, the ratio of fibers to chips or powders is about 5, 10, 15, 20, 25, 30, 35, 40 or 45 fibers to about 30, 35, 40.45, 50, 55, 60 , 65 or 70 chips.

[00167] In some embodiments, the bone material comprises ATM fibers (DBM) of demineralized bone and demineralized ATM shavings in a ratio of 30:60. In some embodiments, the bone material comprises demineralized bone ATM fibers and demineralized bone ATM chips in a ratio of 25:75 to about 75:25 of fibers for chips.

[00168] In some modalities, the TMJ of demineralized bone can be seeded with collected bone cells and / or bone tissue, such as, for example, cortical bone, autogenous bone, halogenic bones and / or xenogenic bone. In some modalities, the ATM of DBM can be seeded with collected cartilage cells and / or cartilage tissue (for example, autogenous, halogenous and / or xenogenic cartilage tissue). For example, before insertion into the target tissue site, the ATM of DBM can be moistened with graft bone cells / tissues, usually with cells / bone tissue aspirated from the patient, in a ratio of about

Petition 870180049765, of 11/06/2018, p. 79/129 / 108 of 3: 1, 2: 1, 1: 1, 1: 3 or 1: 2 in volume. The cells / bone tissue can penetrate the supplied DBM ATM, and the ATM can be kneaded by hand or machine, thus obtaining a flexible and cohesive consistency that can subsequently be packed into the bone defect. In some embodiments, the DBM ATM provides a malleable, non-water-soluble carrier that allows precise placement and retention at the implant site. In some modalities, the collected bone and / or cartilage cells can be mixed with Oxil33 and sown inside the TMJ.

[00169] In some embodiments, the bone material also comprises mineral particles, such as, for example, ceramics. In some embodiments, the particles in the implant comprise a resorbable ceramic, bone, synthetic degradable polymer, hyaluronic acid, chitosan or combinations thereof. In some embodiments, the particles comprise cortical, spongy and / or cortical-spongy, halogenic, xenogenic or transgenic bone tissue. In some embodiments, mineral particles comprise, consist essentially of or consist of bone powder, demineralized bone powder, porous calcium phosphate ceramic, hydroxyapatite, tricalcium phosphate, bioactive glass or combinations thereof.

[00170] In some embodiments, the bone material may comprise a polycarbonate derived from tyrosine (DTE-co-DT carbide) of resorbable ceramic (for example, hydroxyapatite, tricalcium phosphate, bioglass, calcium sulfate), in which the pendant group through tyrosine - an amino acid - is an ethyl ester (DTE) or free carboxylate (DT) or combinations thereof.

[00171] In some embodiments, the bone material may contain an inorganic material, such as an inorganic ceramic and / or bone substitute material. Inorganic materials or bone substitute materials

Petition 870180049765, of 11/06/2018, p. 80/129 / 108 examples include but are not limited to aragonite, dahlite, calcite, brushite, amorphous calcium carbonate, vaterite, weddellite, whelellite, struvite, urate, ferrihydrate, francolite, monohydrocalcite, magnetite, goetite, dentin , calcium carbonate, calcium sulfate, calcium phosphosilicate, sodium phosphate, calcium aluminate, calcium phosphate, hydroxyapatite, alpha-tricalcium phosphate, dicalcium phosphate, β-tricalcium phosphate, tetracalcium phosphate, amorphous calcium phosphate, octhal phosphate , fluoroapatite BIOGLASS ™, chlorapatite, magnesium-substituted tricalcium phosphate, carbonate hydroxyapatite, substituted forms of hydroxyapatite (for example, bone-derived hydroxyapatite can be replaced by other ions such as phloride, chloride, magnesium, sodium, potassium, etc.) , or combinations or derivatives thereof.

[00172] In some modalities, including inorganic ceramics, such as, for example, calcium phosphate in the implant, these will act as a local source of calcium and phosphate for cells that try to deposit new bone. Inorganic ceramics also provide compression strength and load bearing characteristics for the implant.

[00173] In some embodiments, the mineral particles in the implant comprise tricalcium phosphate and hydroxyapatite in a ratio of about 80:20 to about 90:10. In some embodiments, the mineral particles in the implant comprise tricalcium phosphate and hydroxyapatite in a ratio of about 70:30 to about 95: 5. In some embodiments, the mineral particles in the implant comprise tricalcium phosphate and hydroxyapatite in a ratio of about 85:15.

[00174] In some modalities, the bone material comprises mineral particles that offer resistance to compression. In some embodiments, mineral particles (eg ceramic) comprise at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% , 65%, 70%, 75%, 80%, 85%, 90%, or about 95% by weight of

Petition 870180049765, of 11/06/2018, p. 81/129 / 108

ATM. In some embodiments, the particles are predominantly of any shape (for example, round, spherical, elongated, powders, shavings, fibers, cylinders, etc.). In some embodiments, the bone material comprises mineral particles in an amount of about 0.1% by weight to about 95% by weight of ATM. In some embodiments, the bone material comprises mineral particles in an amount of about 50% by weight to about 80% by weight of ATM. In some modalities, the ATM comprises around 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64 %., 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, or about 79 % by weight of ATM.

[00175] In some embodiments, mineral particles are present in an amount of about 0.1% by weight to about 30% by weight of bone material. In some embodiments, mineral particles are present in an amount between about 0.01% by weight to about 50% by weight of ATM. In some embodiments, mineral particles are present in an amount between about 7.0% by weight to about 50% by weight of ATM. In some embodiments, the mineral particles are present in an amount of about 0.1% by weight to about 10% by weight, about 10% by weight to about 20% by weight, about 20% by weight at about 30% by weight, about 30% by weight to about 40% by weight or about 40% by weight to about 50% by weight.

[00176] In some embodiments, the porosity of the particles comprises from 0 to 50%, in some embodiments, the porosity of the particles comprises from 5% to 25%. In some embodiments, the particles are not entangled with each other, but come into contact with each other and the portions of each particle overlap in the bone material to provide resistance to compression. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the particles overlap each other at the ATM.

Petition 870180049765, of 11/06/2018, p. 82/129 / 108 [00177] In some embodiments, the particles are randomly distributed through the ATM. In other embodiments, the particles are uniformly or evenly distributed throughout the ATM. In some embodiments, the particles can be dispersed in the ATM using a dispersing agent. In other embodiments, the particles can be agitated in the polymer and mechanical agitation will distribute the particles in the ATM until the desired distribution is achieved (for example, random or uniform).

[00178] In some embodiments, the bone material can be mixed with one or more therapeutic agents, for example, an anti-inflammatory agent, an analgesic agent, an osteoinductive growth factor, an antimicrobial agent or a combination thereof. Osteoinductive agents include one or more members of the Bone Morphogenetic Protein ("BMPs") family. BMPs are a class of proteins that are believed to have osteoinductive or growth-promoting activities in endogenous bone tissue, or to function as pro-collagen precursors. Known members of the BMP family include, but are not limited to, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-14 (GDF-5), BMP-15, BMP-16, BMP-17, BMP-18 as well as polynucleotides or polypeptides thereof, as well as polypeptides or mature polynucleotides that encode them.

[00179] BMPs used as osteoinductive agents comprise one or more among BMP-1; BMP-2; BMP-3; BMP-4; BMP-5; BMP-6; BMP-7; BMP-8; BMP-9; BMP-10; BMP-11; BMP-12; BMP-13; BMP-15; BMP-16; BMP-17; or BMP-18; as well as any combination of one or more of these BMPs, including the entire BMPs or fragments thereof, or combinations thereof, such as polypeptides or polynucleotides that encode the polypeptide fragments of all of the BMPs cited. The agents

Petition 870180049765, of 11/06/2018, p. 83/129 / 108 isolated BMP osteoinducers can be administered as polynucleotides, polypeptides, whole protein or combinations thereof. [00180] In effect, osteoinductive factors are recombinant human bone morphogenetic proteins (rhBMPs) because they are available in unlimited quantities and do not transmit infectious diseases. In some embodiments, the bone morphogenetic protein is rhBMP-2, rhBMP-4, rhBMP-7 or their heterodimers.

[00181] Recombinant BMP-2 can be used in a concentration of about 0.4 mg / ml to about 10.0 mg / ml, in some cases about 1.5 mg / ml. However, any bone morphogenetic protein is contemplated including bone morphogenetic proteins designated as BMP-1 through BMP18. BMPs are available in Wyeth, Cambridge, Mass. and BMPs and the genes encoding them can also be prepared by one skilled in the art, as described in US Patent 5,187,076 to Wozney et al .; US patent 5,366,875 to Wozney et al .; US patent 4,877,864 to Wang et al .; US patent 5,108,922 to Wang et al .; US patent 5,116,738 to Wang et al .; US patent 5,013,649 to Wang et al .; US patent 5,106,748 to Wozney et al .; PCT patents W093 / 00432 to Wozney et al .; WO94 / 26893 to Celeste et al .; and WO94 / 26892 to Celeste et al. All osteoinductive factors are considered if obtained as above or isolated from the bone. Methods for isolating bone morphogenetic proteins from bone are described, for example, in US Patent 4,294,753 to Urist and Urist et al., 81 PNAS 371, 1984.

[00182] The bone material can include or be mixed with one or more members of the TGF-β superfamily. For example, ATM can include AMH, ARTN, GDF1, GDF10, GDF11, GDF15, GDF2, GDF3, GDF3A, GDF5, GDF6, GDF7, GDF8, GDF9, GDNF, INHA, INHBA, INHBB, INHY, INHY, INHY , MSTN, NODAL, NRTN, PSPN, TGFB1, TGFB2, TGFB3, FGF, basic FGF, VEGF, growth factor

Petition 870180049765, of 11/06/2018, p. 84/129 / 108 similar to insulin, EGF, PDGF, nerve growth factor or combinations thereof.

[00183] Bone material can include or be mixed with a therapeutic agent including, but not limited to, IL-1 inhibitors, such as Kineret® (anakinra), which is a non-glycosylated recombinant form of the interleukin receptor antagonist -1 human (IL-1Ra), or AMG 108, which is a monoclonal antibody that blocks the action of IL-1. The bone material can include or be mixed with therapeutic agents including excitatory amino acids such as glutamate and aspartate, antagonists or inhibitors of glutamate binding to NMDA receptors, AMPA receptors and / or kainate receptors. Bone material can include or be mixed with therapeutic agents to reduce inflammation, including but not limited to, interleukin-1 receptor antagonists, thalidomide (a TNF-α release inhibitor), thalidomide analogs (which reduce the production of TNF-α by macrophages), quinapril (an inhibitor of angiotensin II, which overregulate TNF-α), interferons such as IL-11 (which modulates the expression of the TNF-α receptor), or aurin-tricarboxylic acid (which inhibits TNF-a).

[00184] The bone material may include or be mixed with a therapeutic agent including, but not limited to, an analgesic agent. Examples of analgesic agents include, but are not limited to, acetaminophen, tramadol, lidocaine, bupivacaine, ropivacaine, opioid analgesics such as buprenorphine, butorphanol, dextromoramide, dezocine, dextropropoxyphene, diamorphine, fentanyl, hydenthanone, alfentanil, hydentanyl, sufentanil, hentanidone, alfentanil, hentanidone, alfentanil, hydentanine, alfentanil, hydentanine, alfentanil levomethadil, levorfanol, meperidine, methadone, morphine, nalbuphine, opium, oxycodone, papaveretum, pentazocine, pethidine, phenoperidine, piritramide, dextropropoxyphene, remifentanil, sufentanil, tilidine, tramadol, codeine, dihidrocodine, teninhydrocode of the same.

Petition 870180049765, of 11/06/2018, p. 85/129 / 108 [00185] Bone material may include or be mixed with a therapeutic agent including, but not limited to, an anti-inflammatory agent. An example of an anti-inflammatory agent includes, but is not limited to, clonidine, sulindac, sulfasalazine, naroxin, diclofenac, indomethacin, ibuprofen, flurbiprofen, ketoprofen, aclofenac, alloxyprine, approx., Aspirin, diflunisal, phenphenoxide, mefenamic acid, mefenamic acid , phenylbutazone, piroxicam, meloxicam, salicylamide, salicylic acid, esoxisulindac, tenoxicam, ketorolac, clonidine, flufenisal, salsalate, triethanolamine salicylate, aminopyrine, antipyrin, oxyphenbutazone, acid, flixone, flixone, cintazine, cintazine, cintazine colchicine, demecolcine, allopurinol, oxipurinol, benzidamine hydrochloride, dimefadan, indoxol, intrazol, mimeban hydrochloride, paranylene hydrochloride, tetridamine, benzindopyrine hydrochloride, fluprofen, ibufenac, naproxol, phenbufen, cinchofamide, difinamide, cinchofenamide , letimide hydrochloride, nexeridine hydrochloride, octazamide, molinazole, neocinchophene, neem azole, proxazole citrate, tesmetam tolmetin, triflumidate, fenamates (mefenic acid, meclophenic acid), nabumetone, celecoxib, etodolac, nimesulide, apazone, gold, tepoxaline; dithiocarbamate, or a combination thereof.

[00186] Anti-inflammatory agents also include steroids, such as, for example, 21-acetoxypregnenolone, alclomethasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, chlortortone, cloprednol, cortisol, cortisol, cortisol, cortisol, cortisol, cortisol, cortisol, cortisol, desonide, deoximatasone, dexamethasone, dexamethasone 21-acetate, dexamethasone 21-phosphate di-Na salt, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumetasone, flunisolide, fluocinyl fluoride, fluocinolide , fluorometolone, d and fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate,

Petition 870180049765, of 11/06/2018, p. 86/129 / 108 formocortal, halquinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortate, hydrocortisone, loteprednol etabonate, mazipredone, medrisone, meprednisone, methylprednisolone, mometasone furoate, predetone, predetone, predetone, predetone, predetone, predetone, predetone prednisolone sodium, prednisone, prednival, prednilidene, rimexolone, thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide or a combination thereof.

[00187] Bone material may include or be mixed with a therapeutic agent including, but not limited to, a statin. Examples of a useful statin include, but are not limited to, atorvastatin, simvastatin, pravastatin, cerivastatin, mevastatin (see US Patent No. 3,883,140, the full description of which is incorporated by reference), velostatin (see also synvinoline; see US patent 4,448,784 and 4,450,171 whose entire disclosures are hereby incorporated by reference), fluvastatin, lovastatin, rosuvastatin and fluindostatin (Sandoz XU-62-320), dalvastain (Order Publication EP 738510 A2, the full description of which is incorporated herein by reference), eptastatin, pitavastatin or pharmaceutically acceptable salts thereof or a combination thereof. In various embodiments, the statin may comprise mixtures of the (+) R and (-) S enantiomers of the statin. In several embodiments, the statin may comprise a 1: 1 racemic mixture of the statin.

[00188] In some embodiments, the bone material may include an antimicrobial agent. In some embodiments, the antimicrobial agent may include one or more of triclosan, also known as 2,4,4'-trichloro-2'-hydroxydiphenyl ether, chlorhexidine and its salts, including chlorhexidine acetate, chloro-gluconate hexidine, chlorohexidine hydrochloride and chlorhexidine sulfate, silver and its salts, including silver acetate, silver benzoate, silver carbonate, silver citrate, silver iodate, iodide

Petition 870180049765, of 11/06/2018, p. 87/129 / 108 silver, silver lactate, silver laurate, silver nitrate, silver oxide, silver palmitate, silver protein and silver sulfadiazine, polymyxin, tetracycline, aminoglycosides such as tobramycin and gentamicin, rifampin, bacitracin, neomycin, chloramphenicol, miconazole, quinolones such as oxolinic acid, norfloxacin, nalidixic acid, pefloxacin, enoxacin and ciprofloxacin, penicillins like oxacillin and pipracil, nonoxynol 9, fusidic acid, or cephalosporins.

[00189] Examples of antimicrobial agents include, by way of illustration and not limited to, acedapsone; sodium acetosulfone; alamecine; alexidine; amdinocillin; pivoxil amdinocillin; amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacin sulfate; aminosalicylic acid; sodium aminosalicylate; amoxicillin; amphomycin; ampicillin; ampicillin sodium; apalcilin sodium; apramycin; aspartocin; astromycin sulfate; avilamycin; avoparcin; azithromycin; azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin; bacitracin methylene desalicylate; zinc bacitracin; bambermicins; calcium benzoyls; berithromycin; betamycin sulfate; biapenem; biniramycin; biphenamine hydrochloride; bispirithione magsulfex; buticacin; butyrosine sulfate; capreomycin sulfate; carbadox; disodium carbenicillin; carbenicillin indanyl sodium; phenyl sodium carbenicillin; potassium carbenicillin; carumonam sodium; cefaclor; cefadroxil; cefamandol; cefamandole naphate; cephamandole sodium; cefaparol; cephatrizine; cefazaflur sodium; cefazolin; cefazolin sodium; cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol; cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium; monosodium cefonicide; sodium cephalide; cefoperazone sodium; ceforanide; sodium cefotaxime; cefotetan; disodium cefotetan; cefotiam hydrochloride; cefoxitin; cefoxitin sodium; ccfpimizole; ccfpimizole sodium; cefpiramide; ccfpiramide sodium; ccfpiromc sulfate; cefpodoxime proxetil; ccfprozil; cefroxadine; cefsulodin sodium; ceftazidime; ceftibuten;

Petition 870180049765, of 11/06/2018, p. 88/129 / 108 ceftizoxima sodium; ceftriaxone sodium; cefuroxime; cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cefacetril sodium; cephalexin; cephalexin hydrochloride; cephaloglycine; cephaloridine; cephalotin sodium; cefapirin sodium; cefradin; ketocycline hydrochloride; ketophenicol; chloramphenicol; chloramphenicol palmitate; chloramphenicol pantothenate complex; chloramphenicol sodium succinate; chlorhexidine phosphatylate; chloroxylenol; chlortetracycline bisulfate; chlortetracycline hydrochloride; cytoxacin; ciprofloxacin; ciprofloxacin hydrochloride; cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin; clindamycin hydrochloride; clindamycin hydrochloride palmitate; clindamycin phosphate; clofazimine; benzathine cloxacillin; cloxacillin sodium; chlorhexidine, cloxiquine; colistimethate sodium; colistin sulfate; coumermycin; coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone; daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline; denofungin; diaveridine; dicloxacillin; dicloxacillin sodium; dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline; calcium doxycycline; doxycycline fosfatex; doxycycline hydrate; droxacin sodium; enoxacin; epicillin; epitetracycline hydrochloride; erythromycin; erythromycin acistrate; erythromycin stolate; erythromycin ethyl succinate; erythromycin gluceptate; erythromycin lactobionate; erythromycin propionate; erythromycin stearate; ethambutol hydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine; flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin; furazolium chloride; furazolium tartrate; sodium fusidate; fusidic acid; ganciclovir and ganciclovir sodium; gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin; potassium hetacillin; hexedine; ibafloxacin; imipenem; isoconazole; isepamycin; isoniazid; josamycin; kanamycin sulfate; chitasamycin; levofuraltadone; potassium levopropylcilin; lexithromycin; lincomycin; lincomycin hydrochloride; lomefloxacin; lomefloxacin hydrochloride; mesylate

Petition 870180049765, of 11/06/2018, p. 89/129 / 108 lomefloxacin; loracarbef; mafenide; meclocycline; meclocycline sulfosalicylate; potassium megalomycin phosphate; mequidox; meropenem; metacycline; metacycline hydrochloride; methenamine; methenamine hypurate; methenamine mandelate; sodium methicillin; metioprime; metronidazole hydrochloride; metronidazole phosphate; mezlocillin; mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycin hydrochloride; monensin; monensin sodium; nafcillin sodium; sodium nalidixate; nalidixic acid; nataincin; nebramycin; neomycin palmitate; neomycin sulfate; neomycin undecylenate; netylmycin sulfate; neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone; nifurdazil; nifurimide; nifiupirinol; nifurquinazole; nifurtiazole; nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium; ofloxacin; onetoprim; oxacillin and sodium oxacillin; oximonam; oxymonam sodium; oxolinic acid; oxytetracycline; calcium oxytetracycline; oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin; pefloxacin; pefloxacin mesylate; penamecillin; penicillins such as penicillin g benzathine, penicillin g potassium, penicillin g procaine, penicillin g sodium, penicillin v, penicillin v benzatin, penicillin v hydrabamine, and penicillin v potassium; pentizidone sodium; phenyl aminosalicylate; sodium piperacillin; pirbenicillin sodium; sodium pyridicillin; pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillin pamoate; pivampicillin probenate; polymyxin b sulfate; porphyromycin; propicacin; pyrazinamide; zinc pyrithione; quindecamine acetate; quinupristine; racefenicol; ramosplanin; ranimycin; relomycin; repromycin; rifabutin; rifamethane; rifamexil; rifamide; rifampin; rifapentin; rifaximin; rolithetracycline; rolitetracycline nitrate; rosaramycin; rosaramycin butyrate; rosaramycin propionate phosphate; rosaramycin sodium; rosaramycin stearate; rosoxacin; roxarsone; roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin; sarpicillin; scopafungin; sisomycin; sisomycin sulfate; sparfloxacin; hydrochloride

Petition 870180049765, of 11/06/2018, p. 90/129 / 108 of spectinomycin; spiramycin; stalycin hydrochloride; stefimycin; streptomycin sulfate; streptonicozid; sulfabenz; sulfabenzamide; sulfacetamide; sodium sulfacetamide; sulfacytin; sulfadiazine; sodium sulfadiazine; sulfadoxine; sulfalene; sulfamerazine; sulfameter; sulfamethazine; sulfamethizole; sulfamethoxazole; sulfamonometoxine; sulfamoxol; zinc sulfanylate; sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet; sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine; sulfomyxin; sulopenem; sultamricillin; sodium suncilina; talampicillin hydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin; tetracycline; tetracycline hydrochloride; tetracycline phosphate complex; tetroxoprime; thiamphenicol; potassium tifencilin; cresyl sodium ticarcillin; disodium ticarcillin; monosodium ticarcillin; ticlatone; thiodonium chloride; Tobramycin; tobramycin sulfate; tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines; troleandomycin; troespectomycin sulfate; thyrothricin; vancomycin; vancomycin hydrochloride; virginiamycin; or combinations thereof.

[00190] The antimicrobial agent in the bone material can be an antiviral agent that can be mixed with the bone material. Antiviral agents may include, but are not limited to, vidarabine, acyclovir, famciclovir, valacyclovir, ganciclovir, valganciclovir, nucleoside analog reverse transcriptase inhibitors (such as AZT (zidovudine), ddl (didanosine), ddC (zalcitabine), ddC (zalcitabine) stavudine), and 3TC (lamivudine)), nevirapine, delavirdine, proteinase inhibitors (such as saquinavir, ritonavir, indinavir, and nelfinavir), ribavirin, amantadine, rimantadine, neuraminidase inhibitors (such as zanamivir and oseltamivir, ceconametir, and ceconametir, and ceeconametir, ceconametir, and ceconametir, and ceconametir, and ceconametir, and / or interferons.

Expandable Phase [00191] In some embodiments, the implant may comprise a material, such as, for example, a binder or expandable phase, to facilitate the swelling of the implant. The expandable phase comprises polymers that

Petition 870180049765, of 11/06/2018, p. 91/129 / 108 swell in contact with the fluid (for example, saline, water, body fluid), and thus increase the volume of the implant and that keeps the implant even more in position over time.

[00192] In some embodiments, the binder or expandable phase comprises a range of about 0.1% to about 20% based on the total weight of the implant. In some embodiments, the binder or expandable phase comprises a range of about 0.1% to about 10% based on the total weight of the implant. In some embodiments, the binder or expandable phase comprises about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6% , 7%, 8%, 9% or about 10% based on the total weight of the implant.

[00193] In some embodiments, the binder or expandable phase comprises polymers, monomers, starches, gums, poly (amino acids), or a combination thereof, which swell after contact with fluid (water, saline, body fluids). In various embodiments, the amount of swelling may vary from about 5 to about 100 percent, from about 5 to about 40 percent, or from about 5 to about 20 percent. The time to reach maximum swelling may vary depending on the location and desired property of the implant. In practice, the time to achieve maximum swelling can occur within a period of 5 days, 3 days, 2 days or within a period of 24 hours.

[00194] Suitable intumescent material may include, without limitation, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxyethylcellulose, hydroxyethylcellulose and salts thereof, Carbopol, poly (hydroxyethylmethacrylate), poly (methoxyethylmethacrylate), gelatine, poly (meth), poly (meth) (PMMA), methylmethacrylate (MMA), methylmethacrylate (MMA), polyvinyl alcohols, propylene glycol, PEG 200, PEG 300, PEG 400, PEG 500, PEG 550, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1450, PEG 3350, PEG 4500, PEG 8000 or combinations thereof. In some modalities, the

Petition 870180049765, of 11/06/2018, p. 92/129 / 108 binder or expandable phase includes gelling polymers including, but not limited to, cellulosic polymers, vinyl polymers, such as polyvinylpyrrolidone; acrylic polymers and copolymers, such as acrylic acid polymer, methacrylic acid copolymers, ethyl acrylate-methyl methacrylate copolymers or the like; or mixtures thereof.

[00195] In various implementations, the implant described in the present application further comprises excipients, diluents, biodegradable polymers, intumescent agents, growth factors, antibiotics, solubilizers, stabilizers, bulking agents, antioxidants or binders [00196] A non-limiting list of intumescible materials that the binder or expandable phase may comprise include polyvinyl alcohol (PVA), PVA modified with hydrophilic comonomers, for example, AMPS, the PVA modified with fast cross-linking groups, for example, NAAADA, polyvinylpyrroline-modified PVA (PVP), carboxymethylcellulose, polyethylene glycol (PEG), poly (vinyl ether), PVA and PEG copolymers, polypropylene glycol (PPG), PEG and PPG copolymers, PVA or PPG copolymers, polyacrylonitrile, hydrocolloids, for example, agar, alginates, collagen , elastin, chitin, chitosan, gelatin, sugar, mannitol or the like. In various embodiments, the swelling material includes, for example, poly (N-isopropylacrylamide-co-acrylic acid) -poly (L-lactic acid) (NAL); poly (N-isopropylacrylamide) (PNIPAM) grafted onto other polymers, such as copolymers or polymers of carboxyl cellulose (CMC), including block copolymers and polymers, composites or copolymers with final functionality, containing thermosensitive and poly (2-ethoxy ethyl vinyl ether) and / or poly (hydroxyethyl vinyl ether) and / or (EOVE200-HOVE400), whose sol-gel transition temperature is 20.5 ° C. The intumescent material, in various modalities, can be used to control the release of oxy-cholesterol in the tissue and / or the synovial space.

Petition 870180049765, of 11/06/2018, p. 93/129 / 108 [00197] In some modalities, the ligand or expandable phase includes hyaluronic acid. In some embodiments, the binder or expandable phase includes glycosamino glycans. Non-limiting examples of glycosamino glycans include chondroitin sulfate, dermatan sulfate, keratin sulfate, heparin, heparin sulfate and hyaluronan. In some embodiments, the binder or expandable phase includes mannitol, PEG, magnesium alginate or glycerol.

[00198] The polymers can be cross-linked or slightly cross-linked hydrophilic polymers. Although these polymers can be non-ionic, cationic, zwiterionic or anionic, in various modalities, intumescible polymers are cationic or anionic. In various embodiments, the swelling polymer can contain a multiplicity of acidic functional groups, such as carboxylic acid groups, or salts thereof. Examples of such polymers suitable for use here include those that are prepared from polymerizable acid-containing monomers, or monomers containing functional groups that can be converted to acidic groups after polymerization. Examples of such polymers also include polysaccharide-based polymers such as polymers of carboxymethyl starch and cellulose and poly (amino acids) such as poly (aspartic acid). Some non-acidic monomers can also be included, usually in smaller amounts, in the preparation of the absorbent polymers. Such non-acidic monomers include, for example, monomers containing the following types of functional groups: sulfonate or carboxylate esters, hydroxyl groups, amide groups, amino groups, nitrile groups, quaternary ammonium salt groups and aryl groups (e.g. phenyl, such as those derived from the styrene monomer). Other potential non-acidic monomers include unsaturated hydrocarbons such as ethylene, propylene, 1-butene, butadiene or isoprene.

Petition 870180049765, of 11/06/2018, p. 94/129 / 108 [00199] In some embodiments, the binder or expandable phase comprises substances that are capable of becoming freely permeable after hydration in aqueous fluids. Such substances include polysaccharides, such as gelatin, sucrose, sorbitol, mannans, jaluronic acid, polyamino acids, polyalcohols, polyglycols or the like. In addition to the foregoing, the swellable polymer may also include additional excipients, such as lubricants, flow-promoting agents, plasticizers and non-stick agents. For example, the binder or expandable phase can further include polyethylene glycol, polyvinylpyrrolidone, talc, magnesium stearate, glyceryl beenate, stearic acid or titanium dioxide.

[00200] In several modalities, the particle size distribution of the expandable or binder phase material can be about 10 micrometers, 13 micrometers, 85 micrometers, 100 micrometers, 151 micrometers, 200 micrometers and all the sub-bands between them. In some embodiments, at least 75% of the particles are about 10 micrometers to 200 micrometers in size. In some embodiments, at least 85% of the particles are about 10 micrometers to 200 micrometers in size. In some embodiments, at least 95% of the particles are about 10 micrometers to 200 micrometers in size. In some embodiments, all particles have a size of about 10 micrometers to about 200 micrometers. In some embodiments, at least 75% of the particles are about 20 micrometers to 180 micrometers in size. In some embodiments, at least 85% of the particles have a size of about 20 micrometers to about 180 micrometers. In some embodiments, at least 95% of the particles have a size of about 20 micrometers to about 180 micrometers. In some embodiments, all particles have a size of about 20 micrometers to about 180 micrometers.

Petition 870180049765, of 11/06/2018, p. 95/129 / 108

Cryopreservation and Lyophilization [00201] After decellularization, the tissue can be frozen (for example, cryopreserved) and in other cases, lyophilized or freeze-dried. Before freezing, the tissue can be incubated in a cryopreservation solution. This solution usually contains one or more cryoprotectants to minimize the damage of the ice crystal to the structural ATM that could occur during freezing. Examples of useful cryoprotectants include, without limitation, dimethylsulfoxide (DMSO), dextran, sucrose, 1,2propanediol, glycerol, sorbitol, fructose, trehalose, raffinose, propylene glycol, 2,3 butanediol, hydroxyethyl starch, polyvinylpyrrolidone (PVP), proline ( or other protein stabilizers), human serum albumin or combinations thereof. If the fabric is lyophilized, the solution will usually also contain one or more dry protective components, to minimize structural damage during drying and may include a combination of an organic solvent and water that does not expand or contract during freezing. The cryoprotectants and dry protectors can be the same, or one or more substances. If the tissue is not lyophilized, it can be frozen by placing it (in a sterile container) in a freezer at about -80 ° C, or by immersing it in sterile liquid nitrogen and then storing it at a temperature below -160 ° C until use.

[00202] Any suitable freeze-drying suspension can be used. For example, the freeze-drying suspension medium may comprise water or an aqueous medium, such as saline. In some embodiments, the use of freeze-drying media containing levels of collagen swelling acid or alkali may be included. In other embodiments, the use of freeze-drying suspension media containing levels of acid or alkali collagen swelling may be excluded to prevent damage to the fiber architecture and molecular ultrastructure of the collagen particles. THE

Petition 870180049765, of 11/06/2018, p. 96/129 / 108 For example, freeze-drying suspension media with a pH in the range of about 5 to 12 can be used to suspend cross-linked acellular collagen particles, while for non-cross-linked acellular collagen particles the Freeze-drying suspension can have a pH within the range of about 6 to 8.

[00203] The acellular collagen particles can be suspended in the freeze-drying suspension medium in a range of different concentrations. In several embodiments, the concentration of acellular collagen particles suspended in the freeze-drying suspension medium is in the range of 10 to 80% (w / v), although other concentrations may be used. At the highest end of this range, the amount of acellular collagen particles in the freeze-drying medium has a relatively thick pasty consistency.

[00204] The process can optionally include a step of mixing the suspension of acellular collagen particles in the freeze-drying suspension medium. This ensures an even distribution of the acellular collagen particles in the freeze-drying suspension medium. Treatments of severe mechanical mixing, such as homogenization or blending, should be avoided, however, in order to minimize the damage of the acellular collagen.

[00205] The suspension of acellular collagen particles can, for example, be molded, formed or fused in the desired shape, before lyophilization. In many implementations, the lyophilization suspension medium and the suspended acellular collagen particles are kept in a suitable mold during the freeze drying step. The process can therefore be used for the manufacture of acellular collagenous materials in predetermined ways, which can be easily varied using different molds. Examples of suitable shapes include sheets, plugs, blocks, wedges, beads, strings or variations thereof.

Petition 870180049765, of 11/06/2018, p. 97/129 / 108 [00206] If the ATM is frozen and lyophilized, following incubation in the cryopreservation solution, the tissue can be packaged in a sterile container that is permeable to water vapor, but impermeable to bacteria, for example , a water vapor permeable bag or glass bottle. One side of a useful pouch consists of a medical grade TYVEK® porous membrane, a registered product of the DuPont Company of Wilmington, Dei. This membrane is porous to water vapor and impervious to bacteria and dust. The TYVEK® membrane is thermally sealed to an impermeable sheet of polyethylene laminate, leaving one side open, thus forming a two-sided pouch. The open bag is sterilized by irradiation (for example, γ irradiation) before use. The tissue is placed aseptically (through the open side) in the sterile pouch. The open side is aseptically sealed by heat to close the pouch. The packaged tissue is then protected from microbial contamination through subsequent processing steps.

[00207] In several modalities, crosslinking can be carried out to transmit additional physical resistance to the acellular collagenous material, and an increased resistance to digestive enzymes that may be present in a wound healing environment. Surprisingly, the lyophilized acellular collagenous material formed from cross-linked acellular collagen particles was found to have an improved flexibility compared to the corresponding acellular collagenous material formed from non-cross-linked acellular collagen particles. This is beneficial for the use of acellular collagenous material in the treatment of wounds. Thus, in various embodiments, the acellular collagen particles are cross-linked.

[00208] In various embodiments, crosslinking agents include polyisocyanates, in some cases, diisocyanates. Polyfunctional isocyanates react with amino or hydroxyl groups of different protein chains forming a material that has a stable structure, while

Petition 870180049765, of 11/06/2018, p. 98/129 / 108 retains the original architecture of the acellular collagen and is resistant to enzymatic attack. It is known that antigenicity is associated with the amino groups of the protein chains of the acellular collagen and the reaction of the amino groups with the isocyanate eliminates any antigenicity associated with these groups. In certain respects, diisocyanates include aliphatic, aromatic and alicyclic diisocyanates as exemplified by the 1,6-hexamethylene diisocyanate, toluene diisocyanate, 4,4'-diphenylmethane diisocyanate and 4-diisocyanate , 4'-dicyclohexylmethane, respectively. In other respects, a useful diisocyanate is hexamethylene diisocyanate (HMDI). Carbodiimide crosslinking agents, such as 1-ethyl hydrochloride, can also be used.

3- [3-dimethylaminopropyl] carbodiimide (EDC).

[00209] The extent of crosslinking of the acellular collagen particles can be varied. This usually provides a mechanism to control the rate at which the acellular collagenous material is reabsorbed or degraded during use. Resistance to degradation tends to increase as the extent of crosslinking is increased.

[00210] As an example, the crosslinking of the acellular collagen particles can be performed using HMDI. In certain embodiments, HMDI can be used at a concentration of about 0.01 g to 0.5 g per 50 g of acellular collagen. If the concentration is too high, it can result in reactions of excess crosslinking and foreign bodies. Crosslinking can be carried out over a range of different time periods. As an example, the acellular collagen can be exposed to the cross-linking agent for about 1 hour to about 3 days. Typically, cross-linking is carried out for at least 12 hours, in some cases, at least 20 hours. It will be appreciated that the conditions of crosslinking may routinely vary to adjust the extent of the crosslinking.

[00211] In some embodiments, a lyophilized implant comprises a sterol, for example, an effective amount of Oxil33. A suspension

Petition 870180049765, of 11/06/2018, p. 99/129 / 108 containing an acellular porcine collagen, bone material and oxy-cholesterol is lyophilized or freeze-dried. Any suitable liquid can be used in the aqueous slurry to mix the acellular porcine collagen, bone material and oxy-cholesterol to form the aqueous slurry. Suitable liquids include, for example, aqueous preparations such as water, saline (for example physiological saline), sugar solutions or combinations thereof.

[00212] More particularly, lyophilized implant formulations are typically first prepared as suspensions, then placed on trays, frozen and lyophilized. The total volume of liquid required for pre-lyophilization may be greater than or equal to the amount of liquid required for post-lyophilization.

[00213] The lyophilization process typically includes sublimation of water from a frozen implant formulation under controlled conditions. Freeze drying can be performed using standard equipment as used for freeze drying or vacuum drying. The cycle may vary depending on the equipment and facilities used for filling and finishing.

[00214] Initially, in some embodiments, an aqueous paste containing the implant components, including Oxil33, is placed in molds of desired shapes in a lyophilization chamber under a temperature range and then subjected to temperatures well below the point freezing of the watery paste, usually for several hours. In many cases, the temperature can be less than or equal to -40 ° C for at least 2 hours. After freezing is complete, the freeze-drying chamber and the condenser are evacuated using vacuum pumps, the surface of the condenser having been previously cooled by circulating refrigerant. The condenser would have been cooled below the freezing point of the suspension to about -40 ° C, in some cases to about -50 ° C or less, in other cases to about -60 ° C or less. Additionally, evacuation from the chamber should continue until a

Petition 870180049765, of 11/06/2018, p. 100/129 / 108 pressure of about 50 mTorr to about 600 mTorr, in some cases, about 50 to about 150 mTorr is obtained.

[00215] The lyophilized implant is then heated under vacuum in the chamber and in the condenser. This would usually be accomplished by heating the shelves inside the lyophilizer in which the molds containing the lyophilized implant rest during the lyophilization process to a pressure ranging from about 50 mTorr to about 600 mTorr. The heating process would proceed optimally very gradually, over several hours. For example, the temperature should initially be raised from about -30 ° C to about 10 ° C and maintained for about 10-70 hours. In addition, the lyophilized implant can be raised from the freezing temperature to about 25 ° C to -40 ° C for a period of 30-192 hours. Complete drying can be performed by stabilizing the vacuum, condenser temperature and lyophilized temperature of the implant shelf. After initial drying, the temperature of the lyophilized implant can be increased to about 25 ° C to -40 ° C and maintained for about 5-40 hours. Once the drying cycle is complete, the pressure in the chamber can be slowly released to atmospheric pressure (or slightly below) with sterile dry nitrogen gas (or equivalent gas).

[00216] In some modalities, after lyophilization, the implant is about 95 to about 99.5% free of moisture. In various modalities, the implant is about 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99 or about 99.5% moisture-free. In some embodiments, the implant has about 0.5% to about 5% moisture content after lyophilization. In various modalities, the implant has about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5% moisture content after lyophilization. Lyophilized implants are stable and can be stored over a wide temperature range. Lyophilized implants can be stored at a temperature less than or equal to 30 ° C, for example, refrigerated at 4 ° C or at room temperature (for example, approximately 25 ° C).

Petition 870180049765, of 11/06/2018, p. 101/129 / 108 [00217] The implant is a stable lyophilized implant that is a convenient way to prepare a moldable implant after reconstitution and implant it in or near a bone defect. Oxil33 lyophilized implants are typically reconstituted for use by adding an aqueous solution to rehydrate the lyophilized implant. A wide variety of aqueous solutions can be used to reconstitute a lyophilized implant, for example, physiologically acceptable water, saline, sodium chloride, dextrose, lactated Ringer's solution, phosphate buffered saline (PBS), blood, aspirate bone marrow, bone marrow fractions or a combination thereof.

Preparation Method [00218] In some embodiments, the implant is done by adding an oxy-cholesterol in an amount of about 20% by weight to about 90% by weight for ATM or an acellular porcine collagen, with collagen of acellular porcine in an amount of about 0.1% by weight to about 20% by weight based on the total weight of the implant to form a mixture. In other embodiments, porous ceramic particles are added to the mixture to form the implant, the porous ceramic particles being in an amount of about 30% by weight to about 99.5% by weight based on the total weight of the implant. In some embodiments, the mixture forms an aqueous paste, dough or paste, which can be lyophilized.

[00219] In some embodiments, in the manufacture of the implant, a mixture of the ATM material (for example, acellular collagen and oxy-cholesterol) is combined with the bone material and a liquid to moisten the material to form an aqueous paste, a paste or paste in about 30 seconds. Any suitable liquid can be used including, for example, aqueous preparations such as water, saline (eg physiological saline), sugar solutions, protic organic solvents or liquid polyhydroxy compounds such as glycerol and glycerol esters, or

Petition 870180049765, of 11/06/2018, p. 102/129 / 108 mixtures thereof. The liquid can, for example, make up about 5 to about 70 weight percent of the mixed composition before lyophilization. Once moistened, the implant becomes moldable or flowable in the form of suspension and, in some cases, can be lyophilized.

[00220] In a manufacturing embodiment, a mixture of acellular collagen can be combined with Permacol® paste, an oxy-cholesterol (for example, Oxil33) and a liquid, desirably with an aqueous preparation, to form a moldable cohesive mass. The excess liquid can be removed by any suitable means, including, for example, applying the cohesive mass to a liquid-permeable mold or form and draining the excess liquid.

[00221] In some modalities, the implant is formed by mixing Permacol® paste, acellular collagen and oxy-cholesterol until it forms a coherent mass. In other respects, the bone materials, Permacol® paste and oxy-cholesterol are moistened and mixed in a mixing syringe or similar device.

[00222] In some modalities, the mixture of ATM, bone material and / or oxy-cholesterol are molded to take the shape of the implant. Before, during or after molding, including in some cases applying compression force to the ATM, the acellular porcine collagen can be subjected to one or more additional operations such as heating, lyophilization and / or crosslinking.

[00223] Crosslinking can be used to improve the resistance of the formed ATM. Alternatively, the ATM surface can be cross-linked to reduce the pore size of the porous interior and thereby form the exterior of the ATM which is less permeable and / or less porous than a porous interior. Crosslinking can be achieved, for example, by chemical reaction, by applying energy, such as radiant energy (for example, UV light or

Petition 870180049765, of 11/06/2018, p. 103/129 / 108 microwave energy), drying and / or heating and dye-mediated photo-oxidation, de-hydrothermal treatment, enzymatic treatment or others.

[00224] In some embodiments, a chemical cross-linking agent is used. Examples of suitable crosslinking agents include those that contain reactive bifunctional or multifunctional groups and react with ATM. Chemical crosslinking can be introduced by exposing the ATM material to a chemical crosslinking agent, either by contacting it with a solution of the chemical crosslinking agent, or by exposure to the vapors of the chemical crosslinking agent. This contact or exposure can occur before, during or after a molding operation. In any case, the resulting material can then be washed to remove substantially all remaining amounts from the chemical crosslinker, if necessary or desired, for the performance or acceptance of the final implantable ATM.

[00225] Suitable chemical crosslinking agents include mono- and dialdehydes, including glutaraldehyde and formaldehyde; polyepoxy compounds such as polyglycidyl ethers of glycerol, diglycidyl ethers of polyethylene glycol and other polyepoxy and glycidyl ethers of diepoxy; tanning agents including polyvalent metal oxides such as titanium dioxide, chromium dioxide, aluminum dioxide, zirconium salt, as well as organic tannins and other plant-derived phenolic oxides; chemicals for esterification or carboxyl groups followed by reaction with hydrazide to form acylazide functionalities activated in collagen; dicyclohexylcarbodiimide and its derivatives, as well as other heterobifunctional cross-linking agents; hexamethylene diisocyanate (HMID); and / or sugars, including glucose, will also crosslink ATM material.

[00226] In some embodiments, the matrices are formed by mixing the oxy-cholesterol with an aqueous polymer paste, such as acellular collagen and pouring the mixture into a shape mold. The mixture of

Petition 870180049765, of 11/06/2018, p. 104/129 / 108 composite is freeze-dried and eventually chemically cross-linked and cut to the desired final shape, in some cases, into strips.

[00227] In some embodiments, the oxy-cholesterol is solubilized and mixed with the acellular porcine collagen present in the Permacol® paste. In some embodiments, oxy-cholesterol is solubilized in an organic solvent (for example, ethanol) or optionally with a co-solvent (for example, PEG). Suitable examples of organic solvents include tetrahydrofuran, diethyl ether, dichloromethane, ethyl acetate, acetone, η, η-dimethylformamide, acetonitrile, dimethylsulfoxide, ammonia, tbutanol, n-propanol, ethanol, methanol, acetic acid or combinations thereof. In some embodiments, oxy-cholesterol can be mixed in a ratio of 25:75 to 75:25 with the solvent. In some embodiments, the oxy-cholesterol is mixed in a 50:50 ratio with the solvent to form an oxy-cholesterol solution or suspension, which can then be added to the Permacol® paste and / or bone materials.

[00228] For example, if oxy-cholesterol is Oxil33, it is typically only slightly soluble or insoluble in water. Oxil33 can be solubilized in a non-aqueous solvent, such as, for example, ethanol and mixed in a solution or suspension. In some embodiments, Oxil33 can be filtered by passing Oxil33 and the solvent through a filter (for example, 0.22 microns). This will remove bacteria and other larger particles from the composition. The Oxil33 used in the composition can be micronized before being mixed with solvents and other excipients. In various embodiments, the particle size of Oxil33 can vary from about 1 micron to 250 microns. In some embodiments, Oxil33 can have a particle size of about 5 microns to about 100 microns or about 20 to 50 microns. The Oxil33 in the solution or suspension can be added to an aqueous paste of acellular porcine collagen and / or ceramic particles, mixed, and then dried.

Petition 870180049765, of 11/06/2018, p. 105/129 / 108 [00229] The implant can be used to repair bone and / or cartilage at a target tissue site, for example, one resulting from injury, defect caused during surgery, infection, malignancy or developmental malformation . The implant can be used in a wide variety of orthopedic, periodontal, neurosurgical, oral and maxillofacial surgical procedures, such as the repair of simple and / or composite fractures and / or non-joints; external and / or internal fixations; joint reconstructions, such as arthrodesis; general arthroplasty; hip cup arthroplasty; replacement of the femoral and humeral head; replacement of the surface of the femoral head and / or total replacement of the joint; spinal repairs, including spinal fusion and internal fixation; tumor surgery, for example, defective filling; discectomy; laminactomy; excision of tumors of the spinal cord; anterior cervical and thoracic operations; repairs of spinal injuries; scoliosis, lordosis and kyphosis; intermaxillary fixation of fractures; mentplasty; replacement of the temporomandibular joint; enlargement and reconstruction of the alveolar crest; embedded implantable matrices; implant placement and overhaul; sinus lifts; and / or aesthetic procedures. Specific bones that can be repaired or replaced by implantable TMJ include the ethmoidal, frontal, nasal, occipital, parietal, temporal, mandible, maxilla, zygomatic bone, cervical vertebra, thoracic vertebra, lumbar vertebra, sacrum, rib, sternum, clavicle, scapula , humerus, radius, ulna, carpal bones, metacarpal bones, phalanges, ilium, ischium, pubis, femur, tibia, fibula, patella, calcaneus, tarsal and / or metatarsal bones.

Application of Oxy-cholesterol to ATM [00230] In some modalities, a therapeutic agent (for example, oxy-cholesterol, with or without one or more growth factors) can be placed on the implant or manually, mixing, spraying, impregnating, injecting, brushing and / or pouring to infuse the implant.

Petition 870180049765, of 11/06/2018, p. 106/129 / 108 [00231] The application of oxy-cholesterol to the implant can occur at the time of surgery, by the manufacturer or in any other appropriate way. For example, oxycholesterol evenly dispersed within the Permacol® paste can be placed in a syringe for application to a surgical site. In other respects, a lyophilized mixture of Permacol® and oxy-cholesterol can be reconstituted from a syringe and the syringe can be placed inside the implant by inserting a needle or cannula (piercing the ATM) and injecting the oxy-cholesterol. so that it is evenly distributed throughout the porous interior of the ATM.

[00232] In some modalities, oxy-cholesterol can be applied to the ATM before combining the materials and forming it in the final shape of the implant. In fact, oxy-cholesterol can be mixed into the natural or synthetic polymer (eg, acellular collagen, in some respects, cross-linked) and poured into molds of the final shape of the implant. Alternatively, oxy-cholesterol, like Oxil33, can be applied on and / or on the porous charged ATM after forming it in the final form by mixing, dipping, injection, spraying or a combination thereof.

[00233] The growth factors and oxy-cholesterol of the present application can be eliminated at ATM or with other therapeutic agents. For example, the growth factor can be placed on or in the carrier by electrospray, spraying or ionization impregnation, vibratory dispersion (including sonication), nozzle spraying, compressed air assisted spraying, brushing and / or spillage.

[00234] In some modalities, the interior of the implant is loaded with oxy-cholesterol that works as an osteoinductive factor. In other respects, oxy-cholesterol can be disposed of in a bottle and then a surgeon can mix a fluid with oxy-cholesterol, which can be used to load the Permacol® paste before application to a bone defect site.

Petition 870180049765, of 11/06/2018, p. 107/129 / 108 [00235] The amount of oxy-cholesterol may be sufficient to cause the growth of bones and / or cartilage. In some embodiments, the oxy-cholesterol is Oxil33 and is contained in one or more ATMs in an amount of 1 to 2 mg per cubic centimeter of the ATM.

[00236] In some embodiments, oxy-cholesterol is supplied in a liquid carrier (for example, an aqueous buffered solution or organic solvent). Exemplary aqueous buffered solutions include, but are not limited to, TE, HEPES (2- [4- (2-hydroxyethyl) -liperiperinin] ethanesulfonic acid), MES (2-morpholinoethanesulfonic acid), sodium acetate buffer, sodium citrate, sodium phosphate buffer, a Tris buffer (eg Tris-HCl), phosphate buffered saline (PBS), sodium phosphate, potassium phosphate, sodium chloride, potassium chloride, glycerol, chloride calcium or a combination thereof. In various embodiments, the buffer concentration can be from about 1 mM to 100 mM. In some embodiments, oxy-cholesterol is supplied in a carrier (including a buffer) containing sucrose, glycine, Glutamic acid, sodium chloride and / or pollssorbate 80. Exemplifying organic solvents or non-aqueous solvents include DMSO, acetic acid, acetone, DME , DMF, MTBE, acetonitrile, butanol, butanone, butyl alcohol, ethanol, polyethylene glycol, methanol, chlorobenzene, chloroform, toluene, propanol, pentane, heptane, ethanol, diethyl ether or the like.

[00237] In some embodiments, the oxy-cholesterol disposed in the acellular tissue matrix is OXI133 and comprises Polymorphic Form A which produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 6.1, 12.3 , 16.4, 17.91, 18.6 and 20.94 ± 0.2 degrees 2Θ before being placed in the matrix; or Form B that produces a powder X-ray diffraction pattern comprising one or more of the following reflections: 5.9, 11.9, 13.3, 16.1, 17.96 and 18.82 ± 0.2 degrees 2Θ before being placed in the matrix; or a mixture of them.

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100/108

Treatment Method [00238] In some embodiments, the implant comprises an acellular porcine collagen, in some cases, a cross-linked acellular porcine collagen and an oxy-cholesterol, such as, for example, Oxil33 monohydrate, to promote osteogenesis. In other embodiments, the implant may also include bone material and a fluid. In use, Oxil33 provides therapeutic treatment for bone conditions. Oxil33 facilitates bone formation, osteoblastic differentiation, osteomorphogenesis and / or osteoproliferation. The implant can be administered to treat open fractures and fractures with a high risk of non-union and in individuals with spinal disorders. That is, Oxil33 can induce spinal fusion and can help treat degenerative disc diseases or arthritis that affect the lumbar or cervical vertebrae.

[00239] In some embodiments, the implant, after being lyophilized, is administered by moistening it to impart malleability and moldability properties to the implant. The implant can be molded in different sizes, shapes and configurations. In several modalities, the moldable implant, which comprises an ATM and an oxy-cholesterol, can be placed inside a syringe so that a surgeon can apply it to a surgery site, and / or the syringe containing the implant is still placed in a vacuum bag for additional storage before use. In other cases, the implant is lyophilized and placed in a vacuum sealed pouch for conservation before hydration and use by a surgeon to treat a bone defect. Whether in a syringe or vacuum packaged, these implants can be stored at around 40 ° C and 75% ambient humidity.

[00240] There are several factors that can be taken into account in determining the size, shape and configuration of the implant. For example, both size and shape can allow for ease of

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101/108 implant placement in the target tissue location that is selected. In addition, the shape and size of the implant should be selected to minimize or prevent the implant from moving after the implant. In various embodiments, the implant may be in the form of a rod or a flat surface, such as a film or sheet (e.g., ribbon type) or the like. Flexibility can be a consideration to facilitate placement of the device.

[00241] Mesenchymal stem cells treated with Oxil33 increased osteoblast differentiation. Thus, in some modalities, an ATM comprising Oxil33 can be implanted in a spinal site with mesenchymal stem cells to induce bone growth through differentiation of osteoblasts. Periosteum is a type of tissue that is involved early during a normal bone fracture repair process and can recruit various types of cells (for example, mesenchymal stem cells) and bone growth factors necessary for bone fracture repair. Thus, in some modalities, the periosteum tissue is used as a source of mesenchymal stem cells and / or growth factors in a demineralized bone composition.

[00242] In some embodiments, an implant comprising Oxil33 can be implanted or injected directly into a surgical site in a patient. In some embodiments, the implant is configured to release Oxil33 in the form of a deposit. In some embodiments, a plurality of matrices is provided (for example, in a kit) and administered to a surgical site that triangulates and / or surrounds the site that requires bone growth. In various embodiments, a plurality of matrices comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 matrices. In some embodiments, a plasticizer is used to reduce the glass transition temperature in order to affect implant stability.

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102/108 [00243] Radiographic markers can be included in the implant to allow the user to accurately position it at the patient's target site. These radiographic markers will also allow the user to track the movement and degradation of the implant at the site over time. In this modality, the user can precisely position the implant in place using any of the numerous diagnostic imaging procedures. Such diagnostic imaging procedures include, for example, X-ray imaging or fluoroscopy. Examples of such radiographic markers include, but are not limited to, ceramics, barium, phosphate, bismuth, iodine, tantalum, tungsten and / or metal spheres or particles. In several embodiments, the radiographic marker may have a spherical shape or a ring around the implant. Ceramics in the composition can also be used as a radiographic marker.

[00244] In some modalities, the implant comprising oxycholesterol uniformly disposed in an ATM can be administered to the target site, being shaped according to the needs of a medical procedure and passed through a "cannula" or "needle" that can be a part of an application device, for example, a syringe, a pistol application device, or any medical device suitable for applying the implant to a target organ or anatomical region. The cannula or needle of the device is designed to cause minimal physical and psychological trauma to the patient.

Sterilization [00245] Acellular porcine collagen, bone material, oxy-cholesterol and devices to administer the implant can be sterilized. In various embodiments, one or more components of the ATM, and / or the medical device used to administer the matrix can be sterilized by radiation, in a terminal sterilization step in the final packaging. Terminal sterilization of a product provides a greater guarantee of sterility than of

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103/108 processes as an aseptic process, which requires the individual components of the product to be sterilized separately and the final packaging to be assembled in a sterile environment.

[00246] Typically, in various modalities, gamma radiation is used in the terminal sterilization stage, which involves the use of ionization energy from gamma rays that penetrates deeply into the device. Gamma rays are highly effective in eliminating microorganisms, do not leave residues or have enough energy to transmit radioactivity to the device. Gamma rays can be used when the device is in the package and gamma sterilization does not require high pressures or vacuum conditions, so the package seals and other components are not stressed. In addition, gamma radiation eliminates the need for permeable packaging materials.

[00247] In some modalities, the implant can be packaged in a moisture-resistant package and then terminally sterilized by gamma irradiation. In use, the surgeon removes one or all components from the sterile package for use.

[00248] In several modalities, electron beam radiation (ebeam) can be used to sterilize one or more components of the ATM. The electron beam radiation comprises a form of ionizing energy, which is generally characterized by low penetration and high dose rates. Irradiation with electron beams is similar to gamma processing, as it alters various chemical and molecular bonds in contact, including the reproductive cells of microorganisms. The beams produced for sterilization by electron beams are concentrated and highly charged electron flows, generated by the acceleration and conversion of electricity.

[00249] Other methods can also be used to sterilize the implantable ATM and / or one or more components of the ATM, including, but not limited to,

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104/108 limited to, gas sterilization, such as with ethylene oxide or steam sterilization.

Kits [00250] In several modalities, a kit is provided comprising oxy-cholesterol (for example, Oxil33), ATM (for example, Permacol® paste), bone material, and / or diluents. The kit can include additional parts along with the implantable ATM combined together to be used to implant the ATM (for example, wipes, needles, syringes, mixing syringe or other mixing device). In some cases, oxy-cholesterol has already been incorporated into the ATM present in the kit. The kit can include ATM with or without oxy-cholesterol in a first compartment. The second compartment can include a bottle, holding the diluent and any other instruments necessary for the localized application. A third compartment can include gloves, covers, dressings and other dressing materials to maintain the sterility of the implantation process, as well as an instruction manual, which can include a graphic that shows how to implant the TMJ after hydration. A fourth compartment can include additional needles and / or sutures. Each tool can be packaged separately in a plastic bag that is sterilized by radiation. A fifth compartment can include an agent for radiographic imaging. A kit cover can include illustrations of the implantation procedure and a clear plastic cover can be placed over the compartments to maintain sterility.

[00251] These and other aspects of the present application will be further appreciated after considering the following Examples, which are intended to illustrate certain particular modalities of the application, but are not intended to limit its scope, as defined by the claims.

EXAMPLES [00252] Permacol® paste, an acellular collagen paste derived from the porcine dermis contains 20% cross-linked acellular collagen and

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105/108 80% saline solution. The collagen component is the decellularized porcine dermis that is cryogenically ground and then cross-linked using hexamethylene diisocyanate (HMDI). Moldable implant formulations containing Oxil33 mixed with Permacol® paste were prepared and used in vertebral fusion studies carried out in rabbits and rats. These compositions were neither lyophilized nor loaded into a syringe. These compositions were applied by implanting them on decorticated transversal processes of the studied rabbits and rats.

Example 1 [00253] In this example, the Permacol® slurry was mixed with Oxil33 monohydrate in various amounts as listed in Table 1, below. The moldable formulations of Permacol® paste and Oxil33 monohydrate were applied in various amounts to single-level posterolateral lumbar fusions in rabbits. The crosslinkable moldable bovine collagen formulations were also applied in the same amounts to rabbits in single-level posterolateral lumbar spine fusions. The fusion rates were determined by manual palpation of the operated spinal segments. The results are summarized in Table 1 below.

Table 1 - Single Level Posterolateral Fusion Study in Rabbit

Oxi dose (mg Oxil33 / cc implant) Melting rate of Permacol® Paste from the Moldable Formulation (%) Melting rate of Reticulated Bovine Collagen in the Moldable Formulation (%) 10 50 20 40 66.7 50 80 100 33.3 160 75 33.3 240 66.6 33.3

From the melting rates listed in Table 1 above, it is evident that the moldable formulation of Permacol® Paste and 80 mg / cc of Oxil33 monohydrate provided the highest melting rate of 100% when implanted in rabbits submitted to a single-level posterolateral lumbar fusion, which was an unexpected result. The merger rates obtained by

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106/108 application of implants based on bovine collagen cross-linked to rabbits in identical procedures were significantly less.

Example 2 [00254] In this example, the Permacol® slurry was mixed with Oxil33 monohydrate in various amounts, as listed in Table 2, below. The moldable formulations of Permacol® Paste and Oxil33 monohydrate were applied in various amounts to rats during two-level posterolateral lumbar spine fusions. Moldable formulations with cross-linked bovine collagen were also applied in the same amounts as that of rats during two-level posterolateral lumbar spine fusions. Fusion rates were determined by manual palpation of the operated segments of the column. The results are summarized in Table 2.

Table 2 - Posterior-lateral Fusion Study of 2 Levels of Rat

Oxi dose (Oxil33 mg / cc implant) Melting rate of Permacol Paste in the Moldable Formulation (%) Melting rate of Reticulated Bovine Collagen in the Moldable Formulation (%) 0 0 0 5 0 10 10 60 80 20 90 90 40 100 90 80 100 90

From the melting rates listed in Table 2 above, it is evident that the moldable formulations of Permacol® Paste and both within 40 mg / cm and 80 mg / cc of Oxil33 monohydrate, respectively, provided the melting rate higher than 100% when injected into rats during two-level posterolateral lumbar spine (PLF) fusions. The fusion rates obtained by implants containing cross-linked bovine collagen for the same fusion procedures are significantly lower. These unexpected results were confirmed by X-ray radiographs taken of a rat immediately undergoing a post-operative procedure for a two-level posterolateral lumbar spine fusion as illustrated in FIG. 1 and postoperatively at 4-week and 8-week intervals as illustrated in FIGS. 2 and 3, respectively. FIGS. 1, 2 and 3 represent results of a

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107/108 two-level posterolateral fusion procedure performed on a rat that received a bilateral implant of 0.5 cc of a moldable paste of Permacol® mixed with 40 mg / 1 cc of Oxil33 monohydrate, ceramic granules and sodium carboxylcellulose (CMC) covering the transverse processes L3 to L5 of the rat. In particular, the composition of the moldable paste implant used in this procedure is summarized in Table 3, below.

Table 3 - Composition of the Moldable Implant

description Permacol® Paste (mg) Ceramics (mg) CMC (mg) Oxil33 (mg) 40 mg / cc Oxil33 750 750 20 40

[00255] Compared to the immediate postoperative X-ray radiography of FIG. 1, FIGS. 2 and 3 illustrate the formation of new bone along the lateral margins and trabecular bones inside the implants. The removal of material from the implant was evident in FIGS. 2 and 3 compared to FIG. 1, postoperative radiography. The presence of bridged bones between the transverse L3-L5 process shows that the rat was determined to be radiographically fused.

[00256] FIGS. 4, 5 and 6 are histological slides from rats submitted to posterolateral fusions at two levels. The histological slide of FIG. 4 is the image of a control group of rats that was only a carrier of the implant material without Oxil33. In this image, there is no new bone formation and the fibrous tissue is seen between the residual ceramic granules. FIGS. 5 and 6 are histological slides from rats that have undergone two-level posterolateral fusion containing a moldable implant formulation as in Table 3 above and from which X-ray radiographs after 4 and 8 weeks, respectively, were taken as illustrated in FIGS . 2 and 3. FIGS. 5 and 6 clearly show the disappearance of ceramic granules and an increasing presence of adipose tissue, both indicative of bone formation and confirming the results of FIGS. 2 and 3.

[00257] All patent and non-patent publications cited in this disclosure are hereby incorporated as if each of these

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108/108 publications, patent and non-patent, were incorporated herein by reference in their entirety. In addition, even if the disclosure here has been described with reference to particular examples and modalities, it should be understood that these examples and modalities are merely illustrative of the principles and applications of the present disclosure. Therefore, it should be understood that numerous changes can be made to the illustrative modalities and that other configurations can be designed without departing from the spirit and scope of the present disclosure as defined by the following claims.

[00258] It will be evident to those skilled in the art that various modifications and variations can be made in the various modalities described here without departing from the spirit or scope of the teachings presented here. Thus, it is intended that various modalities cover other modifications and variations of various modalities within the scope of the present teachings.

Claims (20)

1. Implant configured to fit, or close to, a bone defect to promote bone growth, the implant characterized by the fact that it comprises an oxy-cholesterol uniformly disposed in an acellular tissue matrix. 2. Implant according to claim 1, characterized by the fact that (i) the implant is a gel, paste or moldable mass; (ii) the implant is lyophilized; (iii) the implant is lyophilized and placed in a syringe; or (iv) the implant is hydrated and placed in a syringe. 3. Implant according to claim 1, characterized by the fact that (i) oxy-cholesterol comprises (3S, 5S, 6S, 8R, 9S, 10R, 13S, 14S, 17S) 17 - ((S) -2-hydroxyoctan -2-yl) -10, 13-dimethylhexadecahydro-1Hcyclopenta [a] phenanthrene-3,6-diol (Oxil33), Oxil49, Oxil53, Oxil54, Oxil55 or hydrates, solvates, amorphous forms or pharmaceutically acceptable salts of themselves; (ii) the acellular tissue matrix is obtained from a human, porcine, caprine, bovine, ovine or equine source; (iii) the matrix of acellular tissue is porcine collagen; or (iv) the acellular tissue matrix is cross-linked porcine collagen. 4. Implant according to claim 1, characterized in that the matrix of acellular tissue comprises from about 5% by weight to about 25% by weight of acellular porcine collagen and about 0.01% by weight about 90% by weight of oxycholesterol. 5. Implant according to claim 1, characterized in that the implant additionally comprises a fluid of about 25% by weight to about 50% by weight and bone material in an amount of about 25% by weight at about 75% by weight. 6. Implant according to claim 1, characterized by the fact that the acellular tissue matrix comprises porcine collagen Petition 870180049765, of 11/06/2018, p. 118/129 2/5 which is particulate and has a particle size of about 50 pm to about 500 pm. 7. Implant according to claim 5, characterized by the fact that the bone material comprises (i) demineralized bone matrix, mineralized bone, demineralized bone fibers, demineralized bone chips; (ii) inorganic ceramics comprising tricalcium phosphate, hydroxyapatite, biphasic calcium phosphate, porous calcium phosphate ceramic, hydroxyapatite or a combination thereof; (iii) a particle size of about 125 pm to about 750 pm; (iv) tricalcium phosphate and hydroxyapatite in a ratio of about 70:30 to about 90:10; or (v) tricalcium phosphate and hydroxyapatite in a ratio of about 85:15. 8. Implant according to claim 5, characterized by the fact that the fluid comprises water, saline, DMSO, acetic acid, acetone, DME, DMF, MTBE, acetonitrile, butanol, butanone, t-butyl alcohol, ethanol, polyethylene glycol, methanol, chlorobenzene, chloroform, toluene, propanol, pentane, heptane, ethanol, Capriol-90 ™, diethyl ether or a combination thereof. 9. Implant according to claim 1, characterized by the fact that the acellular tissue matrix is of solid or semi-solid particles of collagenous material that are derived from a natural tissue material and the collagenous material preserves the original fiber architecture and the molecular structure of the natural tissue material from which it is derived, and the collagenous material is substantially free of non-fibrous tissue proteins, glycoproteins, cellular elements, lipids or lipid residues, and the collagenous material is not cytotoxic, and the implant is capable of be used as a component of a paste, gel or solution for injection, and the particles of collagenous material have a particle size within the range of approximately 50 microns to approximately 500 microns. Petition 870180049765, of 11/06/2018, p. 119/129 3/5 10. Implant according to claim 1, characterized in that the acellular tissue matrix is a preparation of collagenous fibrous tissue substantially non-resorbable, non-antigenic, originating in human or animal tissue, which is suitable for homo or heterotransplantation as permanent repair of tissue injuries, the preparation of which preserves the natural structure and original architecture of human or animal tissue, is substantially free of proteins and glycoproteins of non-fibrous tissue, is substantially free of cellular elements, is substantially free of lipids and lipid residues and it is not cytotoxic, in which the preparation is capable, when implanted, of being recolonized by the host cells. 11. Implant according to claim 1, characterized by the fact that the implant is configured to be lyophilized and then rehydrated before implantation in, or close to, a bone defect. 12. Method for preparing an implant, the method characterized by the fact that it comprises mixing a fluid with an oxy-cholesterol in an amount of about 0.01% by weight to about 90% by weight based on a total weight of the implant and an acellular tissue matrix in an amount of about 0.1 wt% to about 20 wt% based on the total weight of the implant. Method for preparing an implant according to claim 12, characterized in that it further comprises (i) subjecting the mixed implant to the fluid for lyophilization to form a lyophilized implant; or (ii) in which the acellular tissue matrix is of acellular porcine collagen or acellular porcine crosslinked collagen. 14. Method for preparing an implant according to claim 12, characterized in that the fluid is water, sodium chloride solution, dextrose, Ringer's solution with Lactate, saline Petition 870180049765, of 11/06/2018, p. 120/129 4/5 phosphate buffered, blood, bone marrow aspirate, platelet-rich plasma, bone marrow fractions or a combination of them in an amount of about 3/4 mL to about 10 mL. Method for preparing an implant according to claim 12, characterized in that the implant additionally comprises a binder or phase-expandable material in an amount of about 2% by weight to about 4% by weight based in the total weight of the implant. 16. Method for preparing an implant according to claim 15, characterized in that the binder or the expandable phase material is carboxyl cellulose, pectin, chitosan or a combination thereof. 17. Method for preparing an implant according to claim 13, characterized in that it additionally comprises (i) placing the implant in a mold before lyophilization; (ii) placing the implant in a syringe; (iii) placing the lyophilized implant in a vacuum sealed bag; or (iv) place the syringe containing the implant in a vacuum sealed bag. 18. Method for preparing an implant according to claim 13, characterized in that the volume of the fluid added to the lyophilized implant comprises from about 3/4 ml to about 10 ml. 19. Method for treating a bone defect in a patient in need of it, the method characterized by the fact that it comprises implanting in, or close to, a bone defect an implant comprising an oxy-cholesterol uniformly dispersed in an acellular tissue matrix. 20. Method for treating a bone defect according to claim 19, characterized in that the implant additionally comprises a ligand or an expandable phase material in a Petition 870180049765, of 11/06/2018, p. 121/129 5/5 amount from about 2% by weight to about 4% by weight based on the total weight of the implant.